Therapeutic approaches for treating Alzheimer disease and related disorders through a modulation of synapse function

ABSTRACT

Compositions and methods for the treatment of Alzheimer&#39;s disease and related disorders. More particularly, disclosed are combined therapies that modulate synapse function for treating the disease.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation of commonly assigned copending U.S.patent application Ser. No. 12/915,722, filed on Oct. 29, 2010, which isa continuation-in-part of PCT Application No. PCT/EP2009/055176, filedon Apr. 29, 2009, which is a non-provisional of U.S. ProvisionalApplication No. 61/048,582, filed on Apr. 29, 2008. The contents of allof the foregoing applications are hereby incorporated by reference intheir entirety.

BACKGROUND

The present invention relates to compositions and methods for thetreatment of Alzheimer's disease (AD) and related disorders.

AD is the prototypic cortical dementia characterized by memory deficittogether with dysphasia (language disorder in which there is animpairment of speech and of comprehension of speech), dyspraxia(disability to coordinate and perform certain purposeful movements andgestures in the absence of motor or sensory impairments) and agnosia(ability to recognize objects, persons, sounds, shapes, or smells)attributable to involvement of the cortical association areas. Specialsymptoms such as spastic paraparesis (weakness affecting the lowerextremities) can also be involved (1-4).

Incidence of Alzheimer disease increases dramatically with the age. ADis at present the most common cause of dementia. It is clinicallycharacterized by a global decline of cognitive function that progressesslowly and leaves end-stage patients bound to bed, incontinent anddependent on custodial care. Death occurs, on average, 9 years afterdiagnosis (5).

The incidence rate of AD increases dramatically with age. United Nationpopulation projections estimate that the number of people older than 80years will approach 370 million by the year 2050. Currently, it isestimated that 50% of people older than age 85 years are afflicted withAD. Therefore, more than 100 million people worldwide will suffer fromdementia in 50 years. The vast number of people requiring constant careand other services will severely affect medical, monetary and humanresources (6).

Memory impairment is the early feature of the disease and involvesepisodic memory (memory for day-today events). Semantic memory (memoryfor verbal and visual meaning) is involved later in the disease. Bycontrast, working memory (short-term memory involving structures andprocesses used for temporarily storing and manipulating information) andprocedural memory (unconscious memory that is long-term memory of skillsand procedure) are preserved until late. As the disease progresses, theadditional features of language impairment, visual perceptual andspatial deficits, agnosias and apraxias emerge.

The classic picture of Alzheimer's disease is sufficientlycharacteristic to allow identification in approximately 80% of cases(7). Nevertheless, clinical heterogeneity does occur and not only isthis important for clinical management but provides further implicationof specific medication treatments for functionally different forms. (8).

The pathological hallmark of AD includes amyloid plaques containingbeta-amyloid (Abeta), neurofibrillary tangles (NFT) containing Tau andneuronal and synaptic dysfunction and loss (9-11). For the last decade,two major hypotheses on the cause of AD have been proposed: the “amyloidcascade hypothesis”, which states that the neurodegenerative process isa series of events triggered by the abnormal processing of the AmyloidPrecursor Protein (APP) (12), and the “neuronal cytoskeletaldegeneration hypothesis” (13), which proposes that cytoskeletal changesare the triggering events. The most widely accepted theory explaining ADprogression remains the amyloid cascade hypothesis (14-16) and ADresearchers have mainly focused on determining the mechanisms underlyingthe toxicity associated with Abeta proteins. On contrary, Tau proteinhas received much less attention from the pharmaceutical industry thanamyloid, because of both fundamental and practical concerns. Moreover,synaptic density change is the pathological lesion that best correlateswith cognitive impairment than the two others. Studies have revealedthat the amyloid pathology appears to progress in aneurotransmitter-specific manner where the cholinergic terminals appearmost vulnerable, followed by the glutamatergic terminals and finally bythe GABAergic terminals (11).

SUMMARY

The purpose of the present invention is to provide new therapeuticapproaches for treating AD and related disorders.

The inventors have identified a molecular pathway which is involved inthe genesis of AD and offers novel targets for development of newtreatments to ameliorate AD and related disorders, particularly for thedevelopment of combination therapies using novel or existing moleculespreviously used in other indications. More particularly, the inventorshave identified several drugs which, alone or in combination(s), caneffectively affect such pathway and represent a new and effectivetherapy for the treatment of AD and related disorders.

The invention therefore provides novel compositions and methods fortreating AD disease and related disorders.

More particularly, the invention relates to compositions suitable fortreating Alzheimer's disease or a related disorder in a subject in needthereof, wherein said compositions comprise a drug that amelioratessynapse function.

A further object of this invention relates to compositions suitable fortreating Alzheimer's disease or a related disorder in a subject in needthereof, wherein said compositions comprise a combination of at leasttwo drugs that ameliorate synapse function, for combined, separate orsequential administration.

More preferably, the drug or drugs that ameliorate synapse function bindto or modulate the activity of a protein encoded by a gene selected fromABAT, ABI1, ABL1, ADORA2A, ADORA2B, AKT, AMPK, ANKRA, APBA1, ARHGAP26,ATG5, BASSOON, BDNF, BECLIN1, BIN1, BK channels (KCNMA1, KCNMB1),CACNA1C, CACNA2D3, CACNA2D4, CADPS2, CALCINEURIN, CALMODULIN, CASK,CASR, CAST, CBL, CDC2, CDC42, CDC42BPB, CDC42EP3, CDH13, CDH2, CDK5,CITRON, CNGB3, CORTACTIN, CRAM, CREB, CRMP, CTNNB1, DAB1, DCC, DEPDC2,DHFR, DLG2, DYN1, DYN3, EDNRA, ENDOPHILIN, EPHA3, EPHBR, EPHEXIN,EPHRINA, EPHRINB, ERBB4, ERK1, ERK2, FES, FYN, GABBR1, GABBR2, GABRA2,GABRG2, GAT1, GLRA1, GEPHYRIN, GIPC1, GIPC2, GLUD1, GRANUPHILIN, GRIA2,GRIA3, GRID1, GRID2, GRIK1, GRIK2, GRIN2B, GRIN3A, GRIP, GRM3, GRM5,GRM6, GRM7, GRM8, HOMER, HTR1B, HTR1D, KALIRIN, KCNA2, KCHIP1, KCHIP2.2,KCND2, KCNJ3, KCNJ12, KTN1, KYNU, LYN, MAML3, MINT1, MUC1, MUNC13,MUNC18A, MYO6, MYOL, NAV1, NBEA, NCAM1, NCK1, NCK2, NETRIN1, NFKB1,NGEF, NGF, NGFR, NIL16, NLGN1, NOC2, NOS1, NOTCH1, NOTCH2, NOTCH3, NPC1,NPC2, NPIST, NRG3, NRP1, NRP2, NRX3, NTF3, NTF5, NWASP, OPCML, OPRK1,PAK6, PAK7, PAR1, PARK2, PDE11A, PDE3A/3B, PDE4A/4B/4D, PI3K, PIAS1,PICALM, PICK1, PIP5K, PKA, PKCA, PLD2, PLEXA1, PP1C, PPFIBP1, PRKG1,PSD95, PTN, PTPRF, PYK2, RAB3B, RABPHILIN, RAC1, RAP1, RAS, RASGRF2,RBPJ, REELIN, RGNEF, RHOA, RHOG, RIM2, RIMS1, RIMS2, ROBO2, ROCK2,RPH3AL, SACM1L, SAPAP, SCN1A, SCN1B, SEC24D, SEMA3A, SEMA3C, SEMA3E,SEMA4C, SIAH1A, SLC12A1, SLC12A2, SLC12A5, SLC1A2, SLC6A1, SLC6A18,SLC9A1, SLIT1, SNAP25, SORBS2, SRC, SRGAP3, STX2, STXBP6, SUM1, SV2C,SYNAPTOJANIN, SYNTAXIN1A, SYT12, TACE, TBR1, TRIO, TRKB, TROMBIN, TSPO,UBE2A, ULK4, UNC13C, UNC5C, VAMP2, VAMP5, VELI, VINCULIN, WASPIP, WAVE,WWOX, YAP, and YES1.

Specific and preferred examples of such drugs include, withoutlimitation, compounds selected from acamprosate, alendronate,alfentanil, amiloride, amlodipine, argatroban, aztreonam, baclofen,buclizine, bumetanide, buprenorphine, lidocaine, chlorzoxazone,cilostazol, cinacalcet, dasatinib, desirudin, dyphylline, eletriptan,ergotamine, flunitrazepam, fosphenytoin, imatinib, ketotifen, milrinone,nitroprusside, pegaptanib, pentazocine, phenobarbital, phenforminpregabalin, propylthiouracil, sulfisoxazole, tadalafil, temazepam,terbinafine, tiagabine, topiramate, triamterene, vigabatrin andzonisamide, or a combination thereof.

In a particular embodiment, the compositions of this invention furthercomprise at least one drug that modulates angiogenesis, for combined,separate or sequential use.

Alternatively, or in addition, the compositions of this invention mayfurther comprise at least one drug that modulates cell stress response,for combined, separate or sequential use.

The compositions of this invention typically further comprise apharmaceutically acceptable carrier or excipient.

A further object of this invention resides in a method of producing adrug for treating Alzheimer's disease or a related disorder, the methodcomprising a step of testing a candidate drug for activity on synapsefunction and selecting candidate drugs that ameliorate synapse function.

The invention also relates to a method of producing a composition fortreating Alzheimer's disease or a related disorder, the methodcomprising preparing a combination of a drug that modulates synapsefunction and a drug that modulates angiogenesis or cell stress response,and formulating said combination of drugs for simultaneous, separate orsequential administration thereof to a subject in need thereof.

The invention further relates to a method of treating Alzheimer'sdisease or a related disorder, the method comprising simultaneously,separately or sequentially administering to a subject in need thereof adrug or a combination of drugs that ameliorate synapse function.

The invention further relates to a method of treating Alzheimer'sdisease or a related disorder, the method comprising simultaneously,separately or sequentially administering to a subject in need thereof adrug that modulates synapse function and a drug that modulatesangiogenesis and/or a drug that modulates cell stress response.

The invention further relates to the use of a drug that amelioratessynapse function for the manufacture of a medicament for treatingAlzheimer's disease or a related disorder.

The invention further relates to the use of a combination of at leasttwo drugs that ameliorate synapse function for the manufacture of amedicament for treating Alzheimer's disease or a related disorder,wherein said at least two drugs are administered together, separately orsequentially.

As discussed in the present application, the above therapies andcombination therapies provide novel and effective approaches fortreating AD in human subjects.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A: Effect of acamprosate on neurite outgrowth in beta-amyloidintoxicated rat primary cortical neuron culture. Aβ₂₅₋₃₅ 20 μM producesa significant intoxication, above 25%, compared to vehicle-treatedneurons. This intoxication is significantly prevented by acamprosate.

:p<0.01; *:p<0.05: significantly different from Abeta₂₅₋₃₅. BilateralStudent's t test.

FIG. 1B: Effect of zonisamide on neurite outgrowth in beta-amyloidintoxicated rat primary cortical neuron culture. Aβ₂₅₋₃₅ 20 μM producesa significant intoxication, above 25%, compared to vehicle-treatedneurons. This intoxication is significantly prevented by zonisamide.

:p<0.00001; ****:p<0.0001: significantly different from Abeta₂₅₋₃₅.Bilateral Student's t test.

FIG. 2: Effect of phenformin on neurite outgrowth in beta-amyloidintoxicated rat primary cortical neuron culture.

:p<0.01: significantly different from vehicle. **:p<0.001: significantlydifferent from Aβ₂₅₋₃₅. Bilateral Student's t test. Aβ₂₅₋₃₅ 20 μMproduces a significant intoxication, above 25%, compared tovehicle-treated neurons. This intoxication is efficiently prevented byBDNF at 10 ng/ml (positive control). The intoxication is alsosignificantly prevented by phenformin.

FIG. 3A: Effect of BDNF pre-treatment on LDH release in human Aβ₁₋₄₂toxicity on rat primary cortical cells. Aβ₁₋₄₂ produces a significantintoxication compared to vehicle-treated neurons. 1 h of BDNF (50 ng/ml)pre-treatment significantly protected the neurons from this amyloidinjury (positive control). *:p<0.05: significantly different fromcontrol (no intoxication);

:p<0.05: significantly different from Amyloid intoxication (ANOVA+DunettPost-Hoc test).

FIG. 3B: Effect of zonisamide pre-treatment on LDH release in humanAβ₁₋₄₂ toxicity on rat primary cortical cells. The intoxication issignificantly prevented by zonisamide (−75%).

:p<0.05: significantly different from Aβ₁₋₄₂ intoxication (ANOVA+DunettPost-Hoc test).

FIG. 3C: Effect of phenformin pre-treatment on LDH release in humanAβ₁₋₄₂ toxicity on rat primary cortical cells. The intoxication issignificantly prevented by phenformin (−56%).

:p<0.05: significantly different from Aβ₁₋₄₂ intoxication (ANOVA+DunettPost-Hoc test).

DETAILED DESCRIPTION

The present invention provides new therapeutic approaches for treatingAD or related disorders. The invention discloses novel use of drugs ordrug combinations which allow an effective correction of such diseasesand may be used for patient treatment.

The term “AD related disorder” designates Alzheimer's disease (AD),senile dementia of AD type (SDAT), Parkinson's disease, Lewis bodydementia, vascular dementia, mild cognitive impairment (MCI),age-associated memory impairment (AAMI) and problem associated withageing, post-encephalitic Parkinsonism, ALS and Down syndrome.

As used herein, “treatment” of a disorder includes the therapy,retardation, or reduction of symptoms provoked by the disorder. The termtreatment includes in particular the control of disease progression andassociated symptoms.

The term “ameliorate”, as it refers to synapse function, includes anyincrease in the synapse function as compared to the existing function inthe subject. Such amelioration may include a restoration, i.e., tonormal levels, or lower increase, which are still sufficient to improvethe patient condition. Such an amelioration can be evaluated or verifiedusing known biological tests, such as described in the experimentalsection.

Also, the designation of specific compounds within the context of thisinvention is meant to include not only the specifically named molecules,but also any pharmaceutically acceptable salt, hydrate, ester, ether,isomers, racemate, conjugates, or pro-drugs thereof.

The term “combination” designates a treatment wherein at least two ormore drugs are co-administered to a subject to cause a biologicaleffect. In a combined therapy according to this invention, the at leasttwo drugs may be administered together or separately, at the same timeor sequentially. Also, the at least two drugs may be administeredthrough different routes and protocols. As a result, although they maybe formulated together, the drugs of a combination may also beformulated separately.

As discussed above, the invention relates to compositions and methodsfor treating Alzheimer's disease or a related disorder in a subject inneed thereof, using a drug or a combination of drugs that amelioratesynapse function.

By a comprehensive integration of experimental data covering results ofcell biology studies, expression profiling experiments and geneticassociation studies, describing different aspects of Alzheimer's diseaseand links existing in cellular signalling and functional pathways, theinventors have uncovered that synapse function represents a importantmechanism which is altered in subjects having AD. Genes located in saidfunctional network and implicated in Alzheimer's disease were selectedby the following criteria:

-   -   (1)—direct interaction with the genes causatively responsible        for familial cases of Alzheimer's disease (APP, ApoE,        presenilins, tau protein),    -   (2)—functional partners of the genes selected by the criterion        (1),    -   (3)—nearest functional partners of the genes selected by the        criterion (2).

Through this process, the inventors were able to establish that thenetwork responsible for synapse dysfunction is a major functionalnetwork affected in Alzheimer's disease.

The inventors have more specifically established that the synaptic lossis a functionally-relevant hallmark of Alzheimer's disease, whichultimately leads to progressive cognitive decline, memory loss anddementia. Importantly, synaptic loss correlates better with cognitivedeficit characterized Alzheimer pathology, compared to other AD-specificcellular lesion markers manifested in development of neurofibrillarytangles or deposition of amyloid plaques. Consequently, synapseorganization and synaptic plasticity represent an important target fortherapeutic interventions in the context of Alzheimer's disease.

APP protein is axonally transported and processed in presynapticterminals, leading to high accumulation of Abeta at synapses. Oligomersof Abeta42 as well as amyloid plaques themselves are important forinhibiting long-term potentiation and are primarily responsible formemory impairment in AD patients.

Our data integration procedure revealed a group of genes, which areimplicated in synaptic distortion in AD and which can be formallyseparated into three main functional groups: proteins participating inorganization of post-synaptic density (“PSD”) and correct nerve signaltransmission at post-synaptic membrane; proteins assuringneurotransmitter release; and proteins involved in axon growth anddevelopmental maturation of synaptic machinery.

In a particular embodiment, the present invention thus relates tocompositions and methods using drugs that ameliorate the activity ofproteins involved in post-synaptic density.

The afferent part of excitatory synapses—Post Synaptic Density—arecomposed of a tightly integrated network of scaffold proteins andneurotransmitter receptors that serve a wide range of cognitivefunctions, including memory formation and learning.

Among genes identified by our analysis, the DLG2 gene encodes MAGUKfamily protein that creates an interface between clusteredmembrane-bound receptors, cell-adhesion molecules and actin-basedcytoskeleton. We also identified a large group of glutamate and growthfactor receptors, which interact directly with the DLG2 protein orDLG2/PSD95 proteins complex at excitatory synapses—namely, ErbB4 andTrkB receptors processed and functionally regulated by presenilin(17-18), ionotrophic glutamate receptors of kainate (GRIK2) and NMDAtypes (GRIN3A, GRIN2B), delta type (GRID1, GRID2) glutamate receptors,and G-protein-coupled metabotropic glutamate receptors (GRM3, GRM7, andGRM8). As well, we identified several effector/modulator proteins thatare involved in downstream signalling of the synaptic receptors—citron,a Rho/Rac effector protein, RASGRF2 linking AMPA receptors to activationof RAS/ERK kinases, PARK2 and YES1 kinase.

Other AD-relevant functionally important genes revealed by our analysisinclude presynaptic γ-Neurexin (NXR3) and postsynaptic neuroligin1(NLGN1) proteins that form functional complex involved in dynamicco-regulation of pre- and postsynaptic membranes at excitatory synapses.

In general, the population of PSD proteins potentially implicated inAlzheimer's disease is enriched by receptors participating inorganization of excitatory glutamatergic synapses; only a few inhibitoryneuronal receptors—GABA(A) and GABA(B)—were detected by our data miningscreen.

In another particular embodiment, the present invention relates tocompositions and methods using drugs that ameliorate the activity ofproteins involved in the regulation of neurotransmitter release,preferably at the pre-synaptic membrane.

The release of neurotransmitters at a restricted and highly specializedactive zone of the presynaptic plasma membrane is triggered by actionpotential and is controlled by combined and opposite actions ofvoltage-dependent, calcium-selective Ca_(v) channels (positivemodulators of neurotransmitter release) and of MaxiK channels, largeconductance, voltage and calcium-sensitive potassium channels (negativemodulators of neurotransmitter release). Both types of channels wereselected by our analysis as pertinent therapeutic targets for treatmentof Alzheimer's disease. Additionally, neurotransmitter release atpresynaptic membrane could be modulated by simultaneous application ofdrugs influencing activity of the PRKG1 kinase and/or presynapticGABA(B) receptors.

Our analysis revealed also a group of proteins involved in structuralorganization of neurotransmitter release machinery, responsible formaturation, docking and fusion of synaptic vesicle with the proteinscomposing an active zone—STX2 and STXBP6 proteins participating insynaptic vesicle fusion, BIN1, RAB3B, UNC13C protein essential for thematuration and priming of synaptic vesicles, and RIMS1/2 scaffoldingproteins. The proteins identified by our analysis represent bothstructural proteins, directly involved in exocytosis/endocytosis andrecycling of synaptic vesicles, and their functional activity-dependentmodulators.

In another particular embodiment, the present invention relates tocompositions and methods using drugs that ameliorate the activity ofproteins involved in the regulation of axon growth and guidance.

Proteins participating in regulation of axon growth and guidance allowneuronal precursor cells and axons to migrate toward proper destinationsto ensure correct location and connectivity; they are also involved indevelopmental maturation of newly established synapses as well asdegradation of axons and synopsis in AD disease. These processes play afundamental role for execution of cognitive functions and seem to beextremely vulnerable to toxic effect of Abeta depositions.

Consecutive steps of axon growth and guidance are tightly controlled bycombined actions of extracellular or membrane-tethered Netrins,Semaphorins, Ephrins, DLL and Slits molecules and their respectivefunctional receptors, most of which were revealed by our data miningapproach. Functional outcomes of activation of most of axon growthreceptors are tightly connected with their ability to differentiallymodulate activity of small GTPases RhoA, Rac1 and Cdc42, with the RhoAGTPase being mainly responsible for neurite retraction and growth conecollapse (19).

Among selected genes, DCC axon guidance Netrin receptor is involved bothin neurons attraction and repulsion, while UNC5C netrin receptor ratherpossesses neuron repulsion activity (20); semaphorins and ephrinsmediate growth cones collapse and repulsion in nervous system duringdevelopment and play an important role in synaptic plasticity in adultCNS (21-25). Slits proteins are involved simultaneously to axonsrepulsion and branching, two tightly linked processes, and modulateactivity of netrin receptors (26). Finally, Notch receptor seems toaffect axon guidance through both RBPJ-dependent and RBPJ-independentABL1/DAB1/TRIO pathway controlling organization of actin cytoskeleton(27).

In the present invention, the inventors propose novel compositions,which can be used to ameliorate synapse function altered in Alzheimer'sdisease and other neurogenerative disorders. In a particular embodiment,the compositions and methods of this invention use drugs that amelioratesynapse function through their interaction with or modulation of onegene or protein as listed above.

More specifically, the compositions of this invention comprise a drug ordrugs that ameliorate synapse function through binding to or modulatingthe activity of a protein encoded by a gene selected from ABAT, ABI1,ABL1, ADORA2A, ADORA2B, AKT, AMPK, ANKRA, APBA1, ARHGAP26, ATG5,BASSOON, BDNF, BECLIN1, BIN1, BK channels (KCNMA1, KCNMB1), CACNA1C,CACNA2D3, CACNA2D4, CADPS2, CALCINEURIN, CALMODULIN, CASK, CASR, CAST,CBL, CDC2, CDC42, CDC42BPB, CDC42EP3, CDH13, CDH2, CDK5, CITRON, CNGB3,CORTACTIN, CRAM, CREB, CRMP, CTNNB1, DAB1, DCC, DEPDC2, DHFR, DLG2,DYN1, DYN3, EDNRA, ENDOPHILIN, EPHA3, EPHBR, EPHEXIN, EPHRINA, EPHRINB,ERBB4, ERK1, ERK2, FES, FYN, GABBR1, GABBR2, GABRA2, GABRG2, GAT1,GLRA1, GEPHYRIN, GIPC1, GIPC2, GLUD1, GRANUPHILIN, GRIA2, GRIA3, GRID1,GRID2, GRIK1, GRIK2, GRIN2B, GRIN3A, GRIP, GRM3, GRM5, GRM6, GRM7, GRM8,HOMER, HTR1B, HTR1D, KALIRIN, KCNA2, KCHIP1, KCHIP2.2, KCND2, KCNJ3,KCNJ12, KTN1, KYNU, LYN, MAML3, MINT1, MUC1, MUNC13, MUNC18A, MYO6,MYOL, NAV1, NBEA, NCAM1, NCK1, NCK2, NETRIN1, NFKB1, NGEF, NGF, NGFR,NIL16, NLGN1, NOC2, NOS1, NOTCH1, NOTCH2, NOTCH3, NPC1, NPC2, NPIST,NRG3, NRP1, NRP2, NRX3, NTF3, NTF5, NWASP, OPCML, OPRK1, PAK6, PAK7,PAR1, PARK2, PDE11A, PDE3A/3B, PDE4A/4B/4D, PI3K, PIAS1, PICALM, PICK1,PIP5K, PKA, PKCA, PLD2, PLEXA1, PP1C, PPFIBP1, PRKG1, PSD95, PTN, PTPRF,PYK2, RAB3B, RABPHILIN, RAC1, RAP1, RAS, RASGRF2, RBPJ, REELIN, RGNEF,RHOA, RHOG, RIM2, RIMS1, RIMS2, ROBO2, ROCK2, RPH3AL, SACM1L, SAPAP,SCN1A, SCN1B, SEC24D, SEMA3A, SEMA3C, SEMA3E, SEMA4C, SIAH1A, SLC12A1,SLC12A2, SLC12A5, SLC1A2, SLC6A1, SLC6A18, SLC9A1, SLIT1, SNAP25,SORBS2, SRC, SRGAP3, STX2, STXBP6, SUM1, SV2C, SYNAPTOJANIN, SYNTAXIN1A,SYT12, TACE, TBR1, TRIO, TRKB, TROMBIN, TSPO, UBE2A, ULK4, UNC13C,UNC5C, VAMP2, VAMP5, VELI, VINCULIN, WASPIP, WAVE, WWOX, YAP, and YES1.

The sequences of all of the above listed genes and proteins areavailable from gene libraries and can be isolated by techniques known inthe art. Furthermore, the activity of these genes and proteins can beassessed by techniques known per se in the art, as discussed in theexperimental section.

The invention further describes drugs that can be used to modulate thesetarget genes and proteins. The invention discloses the identificationand activity of particular drugs which, either alone but preferentiallyin combination(s), modulate the above pathway and may be used to treatsaid diseases. In particular, we identified small molecules whichalready exist in the literature but being used to treat distinctdiseases in human subjects.

In this respect, in a most preferred embodiment, the compositions ofthis invention comprise at least an inhibitor of ABAT (preferably,vigabatrin); and/or an inhibitor of ABL1 (preferably imatinib); and/or amodulator of ADORA2B (preferably dyphylline); and/or a modulator of AMPK(preferably phenformin) and/or an inhibitor of CACNA1C (preferablyamlodipine); and/or an inhibitor of CACNA2D3 (preferably pregabalin);and/or a modulator of CASR (preferably cinacalcet); and/or a modulatorof CNGB3 (preferably amiloride), and/or an inhibitor of DHFR (preferablytriamterene), and/or an inhibitor of EPHA3 (preferably dasatinib),and/or antagonist of EDNRA endothelin receptor (preferablysulfisoxazole), and/or a modulator of GABBR2 and glutamatergic receptors(preferably selected from baclofen and acamprosate), and/or a modulatorof GABRA2 (preferably selected from phenobarbital and aztreonam), and/oran antagonist of GRIK1 (preferably, topiramate), and/or a modulator ofGRIN2B and GRIN3A (preferably, acamprosate), and/or a modulator of HTR1Band HTR1D (preferably selected from ergotamine and eletriptan), and/oran antagonist of KCND2 (preferably, lidocaine), and/or a modulator ofKCNMA1 (preferably, chlorzoxazone), and/or a modulator of NOS1(preferably selected from ketotifen and propylthiouracil), and/or aninhibitor of NRP2 (preferably, pegaptanib), and/or a modulator of OPCML(preferably, alfentanil), and/or a modulator of OPRK1 (preferablyselected from buprenorphine and pentazocine), and/or an inhibitor oftrombin receptor PAR1 (preferably, argatroban), and/or an inhibitor ofPDE11A and PDE4A, PDE5A phosphodiesterases (preferably, tadalafil),and/or an inhibitor of PDE3A/3B and PDE4A/4B phosphodiesterases and anactivator of BK channels (preferably, cilostazol), and/or an inhibitorof PDE4D (preferably, milrinone), and/or an activator of PRKG1(preferably selected from nitroprusside, tadalafil and cilostazol),and/or a modulator of RHOA (preferably selected from alendronate andterbinafine), and/or an inhibitor of sodium channel SCN1A and anactivator of BK channels (preferably, zonisamide), and/or an inhibitorof SCN1A/B (preferably, fosphenytoin), and/or an inhibitor of SLC6A1(preferably, tiagabine), and/or a modulator of SLC9A1 (preferably,buclizine), and/or an inhibitor of SLC12A1 (preferably, bumetanide),and/or an inhibitor of TROMBIN (preferably, desirudin), and/or amodulator of TSPO (preferably selected from flunitrazepam andtemazepam), and/or an inhibitor of YES1 (preferably, dasatinib).

As discussed above, the invention particularly proposes to designcombination therapies to address the mechanisms of AD and relateddisorders. In this respect, examples of most preferred target and drugcombinations are disclosed below.

More preferably, the composition of the invention comprises at least oneof the following combinations of drugs, for combined, separate orsequential administration:

-   -   a modulator of AMPK (preferably, phenformin) and an inhibitor of        sodium channel SCN1A and an activator of BK channels        (preferably, zonisamide),    -   a modulator of AMPK (preferably, phenformin) and a modulator of        GABAergic and glutamatergic receptors (preferably, acamprosate),    -   a modulator of AMPK (preferably, phenformin) and an antagonist        of EDNRA endothelin receptor (preferably, sulfisoxazole),    -   a modulator of GABAergic and glutamatergic receptors        (preferably, acamprosate) and an antagonist of EDNRA endothelin        receptor (preferably, sulfisoxazole),    -   an inhibitor of sodium channel SCN1A and an activator of BK        channels (preferably, zonisamide) and an antagonist of EDNRA        endothelin receptor (preferably, sulfisoxazole).    -   a modulator of GABAergic and glutamatergic receptors        (preferably, acamprosate) and an inhibitor of PDE3A/3B and        PDE4A/4B phosphodiesterases and an activator of BK channels        (preferably, cilostazol),    -   an inhibitor of sodium channel SCN1A and an activator of BK        channels (preferably, zonisamide) and a modulator of adenosine        receptor ADORA2B (preferably, dyphylline),    -   an inhibitor of sodium channel SCN1A and an activator of BK        channels (preferably, zonisamide) and an inhibitor of trombin        receptor PAR1 (preferably, argatroban),    -   a modulator of AMPK (preferably, phenformin) and a modulator of        adenosine receptor ADORA2B (preferably, dyphylline),    -   a modulator of AMPK (preferably, phenformin) and an inhibitor of        PDE3A/3B and PDE4A/4B phosphodiesterases and an activator of BK        channels (preferably, cilostazol),    -   an inhibitor of sodium channel SCN1A and an activator of BK        channels (preferably, zonisamide), a modulator of GABAergic and        glutamatergic receptors (preferably, acamprosate),    -   an inhibitor of PDE11A and PDE4A, PDE5A phosphodiesterases        (preferably, tadalafil) and an inhibitor of PDE3A/3B and        PDE4A/4B phosphodiesterases and an activator of BK channels        (preferably, cilostazol), or    -   an inhibitor of sodium channel SCN1A and an activator of BK        channels (preferably, zonisamide) and an inhibitor of PDE3A/3B        and PDE4A/4B phosphodiesterases and an activator of BK channels        (preferably, cilostazol).

Most preferred examples of compositions of this invention comprise acompound selected from acamprosate, alendronate, alfentanil, amiloride,amlodipine, argatroban, aztreonam, baclofen, buclizine, bumetanide,buprenorphine, lidocaine, chlorzoxazone, cilostazol, cinacalcet,dasatinib, desirudin, dyphylline, eletriptan, ergotamine, flunitrazepam,fosphenytoin, imatinib, ketotifen, milrinone, nitroprusside, pegaptanib,pentazocine, phenformin, phenobarbital, pregabalin, propylthiouracil,sulfisoxazole, tadalafil, temazepam, terbinafine, tiagabine, topiramate,triamterene, vigabatrin and zonisamide, or a combination thereof.

Most preferred examples of combination therapies of this inventioncomprise the combined use of at least the following compounds:

phenformin and zonisamide,

phenformin and acamprosate,

phenformin and sulfisoxazole,

acamprosate and sulfisoxazole,

zonisamide and sulfisoxazole,

acamprosate and zonisamide,

acamprosate and cilostazol,

zonisamide and dyphylline,

zonisamide and argatroban,

phenformin and dyphylline,

phenformin and cilostazol,

tadalafil and cilostazol,

zonisamide and cilostazol,

phenformin and tadalafil, or

zonisamide and terbinafine.

Most preferred compositions of this invention comprise at least onecompound chosen from the group consisting of zonisamide, dyphylline,tadalafil, argatroban, acamprosate, cinacalcet, terbinafine, cilostazol,baclofen, phenformin, amlodipine and sulfisoxazole, or salts or prodrugsor derivatives or sustained release formulations thereof.

In the most preferred embodiment, the compositions according to theinvention, comprise zonisamide, or a salt or a prodrug or a derivativeor a sustained release formulation thereof, for treating Alzheimer'sdisease (AD) in a subject in need thereof.

In a more preferred embodiment, the compositions of the inventioncomprise a combination of at least two compounds chosen from the groupconsisting of zonisamide, dyphylline, tadalafil, argatroban,acamprosate, cinacalcet, terbinafine, cilostazol, baclofen, phenformin,amlodipine and sulfisoxazole, or salts or prodrugs or derivatives orsustained release formulations thereof, for simultaneous, separate orsequential administration.

In another embodiment, the composition according to the inventioncomprises a combination of at least two compounds chosen from the groupconsisting of zonisamide, dyphylline, tadalafil, argatroban,acamprosate, cinacalcet, terbinafine, cilostazol, baclofen, phenformin,amlodipine and sulfisoxazole, or salts or prodrugs or derivatives orsustained release formulations thereof, wherein said compositionameliorates synapse function altered in neurodegenerative disordersselected from the group consisting of Alzheimer's disease (AD),Parkinson's disease (PD), Amyotrophic lateral sclerosis (ALS) andmultiple sclerosis (MS).

In another preferred embodiment, the composition of the inventioncomprises a combination of at least two compounds chosen from the groupconsisting of zonisamide, dyphylline, tadalafil, argatroban,acamprosate, cinacalcet, terbinafine, cilostazol, baclofen, phenformin,amlodipine and sulfisoxazole, or salts or prodrugs or derivatives orsustained release formulations thereof for treating Alzheimer's disease(AD).

Preferably, the composition for treating Alzheimer's disease or arelated disorder in a subject in need thereof, comprises at least one ofthe following drug combinations for combined, separate or sequentialadministration:

phenformin and zonisamide,

phenformin and acamprosate,

phenformin and sulfisoxazole,

acamprosate and sulfisoxazole,

zonisamide and sulfisoxazole,

acamprosate and cilostazol,

acamprosate and zonisamide,

zonisamide and dyphylline,

zonisamide and argatroban,

phenformin and dyphylline,

phenformin and cilostazol,

tadalafil and cilostazol,

zonisamide and cilostazol,

phenformin and tadalafil, or

zonisamide and terbinafine.

Preferred compositions for treating Alzheimer's disease comprisezonisamide in combination with at least one compound chosen from thegroup consisting of dyphylline, tadalafil, argatroban, acamprosate,cinacalcet, terbinafine, cilostazol, baclofen, phenformin, amlodipineand sulfisoxazole, or salts or prodrugs or derivatives or sustainedrelease formulations thereof.

In a most preferred embodiment, the composition according to theinvention comprises at least zonisamide and acamprosate, or salts orprodrugs or derivatives or sustained release formulations thereof, forsimultaneous, separate or sequential administration.

Other preferred compositions for treating Alzheimer's disease comprisephenformin, salts or prodrugs or derivatives or sustained releaseformulations thereof, either alone or in combination with at least onecompound selected from the group of zonisamide, dyphylline, tadalafil,argatroban, acamprosate, cinacalcet, terbinafine, cilostazol, baclofen,amlodipine and sulfisoxazole, or salts or prodrugs or derivatives orsustained release formulations thereof.

In another embodiment, the composition further comprises at least onedrug that modulates synapse function, for combined, separate orsequential use.

Preferably, the additional drug that modulates synapse function isselected from an inhibitor of ABAT (preferably, vigabatrin); and/or aninhibitor of ABL1, (preferably imatinib); and/or a modulator of ADORA2B(preferably dyphylline); and/or a modulator of AMPK (preferably,phenformin) and/or an inhibitor of CACNA1C, (preferably amlodipine);and/or an inhibitor of CACNA2D3, (preferably pregabalin); and/or amodulator of CASR (preferably, cinacalcet); and/or a modulator of CNGB3(preferably, amiloride), and/or an inhibitor of DHFR (preferably,triamterene), and/or an inhibitor of EPHA3 (preferably, dasatinib),and/or antagonist of EDNRA endothelin receptor (preferably,sulfisoxazole), and/or a modulator of GABBR2 and glutamatergic receptors(preferably selected from baclofen and acamprosate), and/or a modulatorof GABRA2 (preferably selected from phenobarbital and aztreonam), and/oran antagonist of GRIK1 (preferably, topiramate), and/or a modulator ofGRIN2B and GRIN3A (preferably, acamprosate), and/or a modulator of HTR1Band HTR1D (preferably selected from ergotamine and eletriptan), and/oran antagonist of KCND2 (preferably, lidocaine), and/or a modulator ofKCNMA1 (preferably, chlorzoxazone), and/or a modulator of NOS1(preferably selected from ketotifen and propylthiouracil), and/or aninhibitor of NRP2 (preferably, pegaptanib), and/or a modulator of OPCML(preferably, alfentanil), and/or a modulator of OPRK1 (preferablyselected from buprenorphine and pentazocine), and/or an inhibitor oftrombin receptor PAR1 (preferably, argatroban), and/or an inhibitor ofPDE11A and PDE4A, PDE5A phosphodiesterases (preferably, tadalafil),and/or an inhibitor of PDE3A/3B and PDE4A/4B phosphodiesterases and anactivator of BK channels (preferably, cilostazol), and/or an inhibitorof PDE4D (preferably, milrinone), and/or an activator of PRKG1(preferably selected from nitroprusside, tadalafil and cilostazol),and/or a modulator of RHOA (preferably selected from alendronate andterbinafine), and/or an inhibitor of sodium channel SCN1A and anactivator of BK channels (preferably, zonisamide), and/or an inhibitorof SCN1A/B (preferably, fosphenytoin), and/or an inhibitor of SLC6A1(preferably, tiagabine), and/or a modulator of SLC9A1 (preferably,buclizine), and/or an inhibitor of SLC12A1 (preferably, bumetanide),and/or an inhibitor of TROMBIN (preferably, desirudin), and/or amodulator of TSPO (preferably selected from flunitrazepam andtemazepam), and/or an inhibitor of YES1 (preferably, dasatinib).

In other embodiments, said additional drug that modulates synapsefunction is selected from the drug or drugs that bind to or modulate theactivity of a protein encoded by a gene selected from: ABAT, ABI1, ABL1,ADORA2A, ADORA2B, AKT, AMPK, ANKRA, APBA1, ARHGAP26, ATG5, BASSOON,BDNF, BECLIN1, BIN1, BK channels (KCNMA1, KCNMB1), CACNA1C, CACNA2D3,CACNA2D4, CADPS2, CALCINEURIN, CALMODULIN, CASK, CASR, CAST, CBL, CDC2,CDC42, CDC42BPB, CDC42EP3, CDH13, CDH2, CDK5, CITRON, CNGB3, CORTACTIN,CRAM, CREB, CRMP, CTNNB1, DAB1, DCC, DEPDC2, DHFR, DLG2, DYN1, DYN3,EDNRA, ENDOPHILIN, EPHA3, EPHBR, EPHEXIN, EPHRINA, EPHRINB, ERBB4, ERK1,ERK2, FES, FYN, GABBR1, GABBR2, GABRA2, GABRG2, GAT1, GLRA1, GEPHYRIN,GIPC1, GIPC2, GLUD1, GRANUPHILIN, GRIA2, GRIA3, GRID1, GRID2, GRIK1,GRIK2, GRIN2B, GRIN3A, GRIP, GRM3, GRM5, GRM6, GRM7, GRM8, HOMER, HTR1B,HTR1D, KALIRIN, KCNA2, KCHIP1, KCHIP2.2, KCND2, KCNJ3, KCNJ12, KTN1,KYNU, LYN, MAML3, MINT1, MUC1, MUNC13, MUNC18A, MYO6, MYOL, NAV1, NBEA,NCAM1, NCK1, NCK2, NETRIN1, NFKB1, NGEF, NGF, NGFR, NIL16, NLGN1, NOC2,NOS1, NOTCH1, NOTCH2, NOTCH3, NPC1, NPC2, NPIST, NRG3, NRP1, NRP2, NRX3,NTF3, NTF5, NWASP, OPCML, OPRK1, PAK6, PAK7, PAR1, PARK2, PDE11A,PDE3A/3B, PDE4A/4B/4D, PI3K, PIAS1, PICALM, PICK1, PIP5K, PKA, PKCA,PLD2, PLEXA1, PP1C, PPFIBP1, PRKG1, PSD95, PTN, PTPRF, PYK2, RAB3B,RABPHILIN, RAC1, RAP1, RAS, RASGRF2, RBPJ, REELIN, RGNEF, RHOA, RHOG,RIM2, RIMS 1, RIMS2, ROBO2, ROCK2, RPH3AL, SACM1L, SAPAP, SCN1A, SCN1B,SEC24D, SEMA3A, SEMA3C, SEMA3E, SEMA4C, SIAH1A, SLC12A1, SLC12A2,SLC12A5, SLC1A2, SLC6A1, SLC6A18, SLC9A1, SLIT1, SNAP25, SORBS2, SRC,SRGAP3, STX2, STXBP6, SUM1, SV2C, SYNAPTOJANIN, SYNTAXIN1A, SYT12, TACE,TBR1, TRIO, TRKB, TROMBIN, TSPO, UBE2A, ULK4, UNC13C, UNC5C, VAMP2,VAMP5, VELI, VINCULIN, WASPIP, WAVE, WWOX, YAP, and YES1.

The inventors have established that the above drugs and drugcombinations provide improved and synergistic biological effect leadingto an effective correction or normalization or functional dysregulationleading to AD and related disorders.

The above named compounds are listed in the following table 1, togetherwith their CAS number. As discussed before, it should be understood thatthe invention encompasses the use of the above compounds as well as anypharmaceutically acceptable salt, hydrate, ester, ether, isomers,racemate, conjugates, or pro-drugs thereof. Prodrugs may be prepared(e.g., by coupling the drug to a suitable carrier) to offer a bettercontrol over the pharmacokinetic parameters of the treatment.

TABLE 1 DRUG NAME CAS NUMBER Acamprosate 77337-76-9 Alendronate66376-36-1 Alfentanil 71195-58-9 Amiloride 2016-88-8 Amlodipine88150-42-9 Argatroban 74863-84-6 Aztreonam 78110-38-0 Baclofen 1134-47-0Balsalazide 80573-04-2 Buclizine 82-95-1 Bumetanide 28395-03-1Buprenorphine 52485-79-7 Lidocaine 137-58-6 Chlorzoxazone 95-25-0Cilostazol 73963-72-1 Cinacalcet 226256-56-0 Dasatinib 302962-49-8Desirudin 120993-53-5 Dyphylline 479-18-5 Eletriptan 143322-58-1Ergotamine 113-15-5 Flunitrazepam 1622-62-4 Fosphenytoin 93390-81-9Imatinib 152459-95-5 Ketotifen 34580-14-8 Milrinone 78415-72-2Nitroprusside 15078-28-1 Pegaptanib 222716-86-1 Pentazocine 359-83-1Phenobarbital 50-06-6 Phenformin 114-86-3 Pregabalin 148553-50-8Propylthiouracil 51-52-5 Sulfisoxazole 127-69-5 Tadalafil 171596-29-5Temazepam 846-50-4 Terbinafine 91161-71-6 Tiagabine 115103-54-3Topiramate 97240-79-4 Triamterene 396-01-0 Vigabatrin 60643-86-9Zonisamide 68291-97-4

Examples of pharmaceutically acceptable salts include pharmaceuticallyacceptable acid addition salts, pharmaceutically acceptable baseaddition salts, pharmaceutically acceptable metal salts, ammonium andalkylated ammonium salts. Acid addition salts include salts of inorganicacids as well as organic acids. Representative examples of suitableinorganic acids include hydrochloric, hydrobromic, hydroiodic,phosphoric, sulfuric, nitric acids and the like. Representative examplesof suitable organic acids include formic, acetic, trichloroacetic,trifluoroacetic, propionic, benzoic, cinnamic, citric, fumaric,glycolic, lactic, maleic, malic, malonic, mandelic, oxalic, picric,pyruvic, salicylic, succinic, methanesulfonic, ethanesulfonic, tartaric,ascorbic, pamoic, bismethylene salicylic, ethanedisulfonic, gluconic,citraconic, aspartic, stearic, palmitic, EDTA, glycolic, p-aminobenzoic,glutamic, benzenesulfonic, p-toluenesulfonic acids, sulphates, nitrates,phosphates, perchlorates, borates, acetates, benzoates,hydroxynaphthoates, glycerophosphates, ketoglutarates and the like.Additional examples of pharmaceutically acceptable inorganic or organicacid addition salts are listed in e.g., J. Pharm. Sci. 1977, 66, 2,which is incorporated herein by reference. Examples of metal saltsinclude lithium, sodium, potassium, magnesium salts and the like.Examples of ammonium and alkylated ammonium salts include ammonium,methylammonium, dimethylammonium, trimethylammonium, ethylammonium,hydroxyethylammonium, diethylammonium, butylammonium,tetramethylammonium salts and the like. Examples of organic basesinclude lysine, arginine, guanidine, diethanolamine, choline and thelike.

Therapy according to the invention may be performed alone or as drugcombination, and/or in conjunction with any other therapy, targeting thesame pathway or having distinct modes of actions. It and may be providedat home, the doctor's office, a clinic, a hospital's outpatientdepartment, or a hospital, so that the doctor can observe the therapy'seffects closely and make any adjustments that are needed.

In a particular embodiment, the compositions of this invention furthercomprise at least one drug that modulates angiogenesis, preferably thatincreases angiogenesis, for combined, separate or sequential use. Morepreferably, said at least one drug that modulates angiogenesis isselected from albuterol, alendronate, ambrisentan, aminocaproic acid,balsalazide, becaplermin, cabergoline, clopidogrel, dihydroergotamine,eplerenone, fenoldopam, fludrocortisone acetate, gemfibrozil,hesperetin, leflunomide, L-histidine, liothyronine, marimastat,meloxicam, mepacrine, methazolamide, methimazole, montelukast,netilmicin, nitroglycerin, phenylbutyrate, pyrimethamine, sunitinib,thiethylperazine, tirofiban, topotecan, vidarabine and warfarin (seetable 2 below).

TABLE 2 DRUG NAME CAS NUMBER Albuterol 18559-94-9 Alendronate 66376-36-1Ambrisentan 177036-94-1 Aminocaproic acid 60-32-2 Balsalazide 80573-04-2Becaplermin 165101-51-9 Cabergoline 81409-90-7 Clopidogrel 113665-84-2Dihydroergotamine 6190-39-2 Eplerenone 107724-20-9 Fenoldopam 67227-57-0Fludrocortisone 127-31-1 Gemfibrozil 25812-30-0 Hesperetin 520-33-2Leflunomide 75706-12-6 L-histidine 71-00-1 Liothyronine 6893-02-3Marimastat 154039-60-8 Meloxicam 71125-38-7 Mepacrine 83-89-6Methazolamide 554-57-4 Methimazole 60-56-0 Montelukast 158966-92-8Netilmicin 56391-56-1 Nitroglycerin 55-63-0 Pyrimethamine 58-14-0 Sodiumphenylbutyrate 1716-12-7 Sunitinib 557795-19-4 Thiethylperazine1420-55-9 Tirofiban 144494-65-5 Topotecan 119413-54-6 Vidarabine24356-66-9 Warfarin 81-81-2

Alternatively, or in addition to the preceding embodiment, thecompositions of this invention may further comprise at least one drugthat modulates cell stress response, preferably that inhibits cellstress response, for combined, separate or sequential use.

The most preferred drugs that modulate cell stress response are selectedfrom arabitol, mannitol, metaraminol, omeprazole, prilocaine, rapamycin,rifabutin, thioguanine and trehalose (see table 3 below).

TABLE 3 DRUG NAME CAS NUMBER Arabitol 488-82-4, 7643-75-6, 6018-27-5Mannitol 69-65-8 Metaraminol 54-49-9 Omeprazole 73590-58-6 Prilocaine721-50-6 Rapamycin 53123-88-9 Rifabutin 72559-06-9 Thioguanine 154-42-7Trehalose 99-20-7

In a particular embodiment, the invention relates to a compositioncomprising a drug that ameliorates synapse function, a drug thatincreases angiogenesis, and a drug that inhibits cell stress response,for simultaneous, separate or sequential administration.

Other therapies used in conjunction with drug(s) or drug(s)combination(s) according to the present invention, may comprise one ormore drug(s) that ameliorate symptoms of Alzheimer's disease or drug(s)that could be used for palliative treatment of Alzheimer's disease.Preferably, said one or more drug(s) is/are selected from 3APS, AAB-001,ABT-089, ABT-126, AC-3933, ACC-001, Acetaminophen, AFFITOPE AD01,AFFITOPE AD02, alpha-lipoic acid, alpha-tocopherol, AN1792, anti-Abeta,AQW051, Aripiprazole, Atomoxetine, Atorvastatin, AVE1625, AVP-923,AZD0328, AZD3480, Bapineuzumab, BAY94-9172 (ZK 6013443), Bifeprunox,Bioperine, BMS-708163, BRL-049653, Bryostatin, CAD106, Celecoxib,CERE-110, Cerebrolysin, CHF 5074, Choline, Circadin, Citalopram,Coenzyme Q, Copper, CTS21166, Curcumin, CX516 (Ampalex), CX717,Cyclophosphamate, DCB-AD1, Dextroamphetamine, DHA (DocosahexaenoicAcid), Digoxin, Dimebon (Latrepirdine), Divalproex, DMXB-A, Donepezil,Doxycycline, Egb 761, EHT 0202 tazolate, ELND005 (scyllo-inositol), EPAX1050TG, Ergoloid mesylate, Epigallocatechin-Gallate, Escitalopram,Estradiol, Estrogen, Etanercept, EVP-6124, EVT101, Exelon, Fish oil,FK962, florpiramine F 18, Folate+Vitamin B6+Vitamin B21, Gabapentin,Galantamine, Gemfibrozil, Ginkgo biloba extracts (for example EGb 761 orCP401), improved extracts of Ginkgo biloba (for example enriched inactive ingredients or lessened in contaminant) or drug containing Ginkgobiloba extracts (for example Tanakan or Gingkor fort), Glucose,L-Glutamic Acid, GSI 136, GSI-953, GSK239512, GSK933776A, Haloperidol,HF0220, Huperzine A, hydrocodone/APAP, Ibuprofen, IFN-alpha2A,Indomethacin, Insulin, Intravenous Immunoglobulin, Ketasyn, Lecozotan,Leuprolide, Levodopa, Lipoic Acid, Lithium, Lorazepam, Lovostatin,Lutein, LY2062430 (solanezumab), LY2811376, LY450139, LY451395,MABT5102A, Malate, Masitinib (AB1010), Medroxyprogesterone, Melatonin,MEM 1003, MEM 3454, Memantine, Methylene blue, Methylphenidate,Mifepristone, MK0249, MK0677, MK0952, MK0952, MK3328, Modafinil,MPC-7869, NADH, Naproxen, Nefiracetam, Neptune Krill Oil, Neramexane,NIC5-15, Nicoderm Patch, Nicotinamide (vitamin B3), Novasoy, NP031112,NS 2330, NSA-789, NSAIDs, Olanzapine, omega-3 polyunsaturated fattyacids (EPA+DHA), ONO-2506PO, Oxybate, Panax Ginseng, PAZ-417, PBT2,Perphenazine, PF-04360365, PF-04447943, PF-04494700, Phenserine,Phosphatidylserine, Pitavastatin, Posiphen, PPI-1019 (APAN),Pravastatin, Prazosin, Prednisone, Progesterone, PRX-03140, PYM50028,Quetiapine, R1450, Raloxifene, Ramipril, Rasagiline, Razadyne,resveratrol, rifampicin, risperidone, Rivastigmine, RN1219, RO5313534,Rofecoxib, Rosiglitazone, Salvia officinalis (sage), SAM-315, SAM-531,SAM-760, SB-742457, Selenium, Sertraline, SGS-742, Simvastatin,SK-PC-B70M, Solanezumab, SR57667B, SRA-333, SRA-444, SSR180711C, ST101,T-817MA, Tacrine, Tarenflurbil, Testosterone, Tramiprosate (3APS),Trazodone, TRx0014 (methylthioninium chloride), Tryptophan, V950,Valproate, Varenicline, Vitamin C, Vitamin E, VP4896, Xaliproden,Zeaxanthin, Zolpidem, and ZT-1 (DEBIO-9902 SR).

The compositions of the invention typically comprise one or severalpharmaceutically acceptable carriers or excipients. The duration of thetherapy depends on the stage of the disease being treated, thecombination used, the age and condition of the patient, and how thepatient responds to the treatment.

The dosage, frequency and mode of administration of each component ofthe combination can be controlled independently. For example, one drugmay be administered orally while the second drug may be administeredintramuscularly. Combination therapy may be given in on-and-off cyclesthat include rest periods so that the patient's body has a chance torecover from any as yet unforeseen side-effects. The drugs may also beformulated together such that one administration delivers all drugs.

The administration of each drug of the combination may be by anysuitable means that results in a concentration of the drug that,combined with the other component, is able to correct the functioning ofpathways implicated in AD.

While it is possible for the active ingredients of the combination to beadministered as the pure chemical it is preferable to present them as apharmaceutical composition, also referred to in this context aspharmaceutical formulation. Possible compositions include those suitablefor oral, rectal, topical (including transdermal, buccal andsublingual), or parenteral (including subcutaneous, intramuscular,intravenous and intradermal) administration.

More commonly these pharmaceutical formulations are prescribed to thepatient in “patient packs” containing a number dosing units or othermeans for administration of metered unit doses for use during a distincttreatment period in a single package, usually a blister pack. Patientpacks have an advantage over traditional prescriptions, where apharmacist divides a patient's supply of a pharmaceutical from a bulksupply, in that the patient always has access to the package insertcontained in the patient pack, normally missing in traditionalprescriptions. The inclusion of a package insert has been shown toimprove patient compliance with the physician's instructions. Thus, theinvention further includes a pharmaceutical formulation, as hereinbefore described, in combination with packaging material suitable forsaid formulations. In such a patient pack the intended use of aformulation for the combination treatment can be inferred byinstructions, facilities, provisions, adaptations and/or other means tohelp using the formulation most suitably for the treatment. Suchmeasures make a patient pack specifically suitable for and adapted foruse for treatment with the combination of the present invention.

The drug may be contained in any appropriate amount in any suitablecarrier substance, and is may be present in an amount of 1-99% by weightof the total weight of the composition. The composition may be providedin a dosage form that is suitable for the oral, parenteral (e.g.,intravenously, intramuscularly), rectal, cutaneous, nasal, vaginal,inhalant, skin (patch), or ocular administration route. Thus, thecomposition may be in the form of, e.g., tablets, capsules, pills,powders, granulates, suspensions, emulsions, solutions, gels includinghydrogels, pastes, ointments, creams, plasters, drenches, osmoticdelivery devices, suppositories, enemas, injectables, implants, sprays,or aerosols.

The pharmaceutical compositions may be formulated according toconventional pharmaceutical practice (see, e.g., Remington: The Scienceand Practice of Pharmacy (20th ed.), ed. A. R. Gennaro, LippincottWilliams & Wilkins, 2000 and Encyclopedia of Pharmaceutical Technology,eds. J. Swarbrick and J. C. Boylan, 1988-1999, Marcel Dekker, New York).

Pharmaceutical compositions according to the invention may be formulatedto release the active drug substantially immediately upon administrationor at any predetermined time or time period after administration.

The controlled release formulations include (i) formulations that createa substantially constant concentration of the drug within the body overan extended period of time; (ii) formulations that after a predeterminedlag time create a substantially constant concentration of the drugwithin the body over an extended period of time; (iii) formulations thatsustain drug action during a predetermined time period by maintaining arelatively, constant, effective drug level in the body with concomitantminimization of undesirable side effects associated with fluctuations inthe plasma level of the active drug substance; (iv) formulations thatlocalize drug action by, e.g., spatial placement of a controlled releasecomposition adjacent to or in the diseased tissue or organ; and (v)formulations that target drug action by using carriers or chemicalderivatives to deliver the drug to a particular target cell type.

Administration of drugs in the form of a controlled release formulationis especially preferred in cases in which the drug, either alone or incombination, has (i) a narrow therapeutic index (i.e., the differencebetween the plasma concentration leading to harmful side effects ortoxic reactions and the plasma concentration leading to a therapeuticeffect is small; in general, the therapeutic index, TI, is defined asthe ratio of median lethal dose (LD50) to median effective dose (ED50));(ii) a narrow absorption window in the gastro-intestinal tract; or (iii)a very short biological half-life so that frequent dosing during a dayis required in order to sustain the plasma level at a therapeutic level.

Any of a number of strategies can be pursued in order to obtaincontrolled release in which the rate of release outweighs the rate ofmetabolism of the drug in question. Controlled release may be obtainedby appropriate selection of various formulation parameters andingredients, including, e.g., various types of controlled releasecompositions and coatings. Thus, the drug is formulated with appropriateexcipients into a pharmaceutical composition that, upon administration,releases the drug in a controlled manner (single or multiple unit tabletor capsule compositions, oil solutions, suspensions, emulsions,microcapsules, microspheres, nanoparticles, patches, and liposomes).

Solid Dosage Forms for Oral Use

Formulations for oral use include tablets containing the activeingredient(s) in a mixture with non-toxic pharmaceutically acceptableexcipients. These excipients may be, for example, inert diluents orfillers (e.g., sucrose, microcrystalline cellulose, starches includingpotato starch, calcium carbonate, sodium chloride, calcium phosphate,calcium sulfate, or sodium phosphate); granulating and disintegratingagents (e.g., cellulose derivatives including microcrystallinecellulose, starches including potato starch, croscarmellose sodium,alginates, or alginic acid); binding agents (e.g., acacia, alginic acid,sodium alginate, gelatin, starch, pregelatinized starch,microcrystalline cellulose, carboxymethylcellulose sodium,methylcellulose, hydroxypropyl methylcellulose, ethylcellulose,polyvinylpyrrolidone, or polyethylene glycol); and lubricating agents,glidants, and antiadhesives (e.g., stearic acid, silicas, or talc).Other pharmaceutically acceptable excipients can be colorants, flavoringagents, plasticizers, humectants, buffering agents, and the like.

The tablets may be uncoated or they may be coated by known techniques,optionally to delay disintegration and absorption in thegastrointestinal tract and thereby providing a sustained action over alonger period. The coating may be adapted to release the active drugsubstance in a predetermined pattern (e.g., in order to achieve acontrolled release formulation) or it may be adapted not to release theactive drug substance until after passage of the stomach (entericcoating). The coating may be a sugar coating, a film coating (e.g.,based on hydroxypropyl methylcellulose, methylcellulose, methylhydroxyethylcellulose, hydroxypropylcellulose, carboxymethylcellulose,acrylate copolymers, polyethylene glycols and/or polyvinylpyrrolidone),or an enteric coating (e.g., based on methacrylic acid copolymer,cellulose acetate phthalate, hydroxypropyl methylcellulose phthalate,hydroxypropyl methylcellulose acetate succinate, polyvinyl acetatephthalate, shellac, and/or ethylcellulose). A time delay material suchas, e.g., glyceryl monostearate or glyceryl distearate may be employed.

The solid tablet compositions may include a coating adapted to protectthe composition from unwanted chemical changes, (e.g., chemicaldegradation prior to the release of the active drug substance). Thecoating may be applied on the solid dosage form in a similar manner asthat described in Encyclopedia of Pharmaceutical Technology.

Several drugs may be mixed together in the tablet, or may bepartitioned. For example, the first drug is contained on the inside ofthe tablet, and the second drug is on the outside, such that asubstantial portion of the second drug is released prior to the releaseof the first drug.

Formulations for oral use may also be presented as chewable tablets, oras hard gelatin capsules wherein the active ingredient is mixed with aninert solid diluent (e.g., potato starch, microcrystalline cellulose,calcium carbonate, calcium phosphate or kaolin), or as soft gelatincapsules wherein the active ingredient is mixed with water or an oilmedium, for example, liquid paraffin, or olive oil. Powders andgranulates may be prepared using the ingredients mentioned above undertablets and capsules in a conventional manner.

Controlled release compositions for oral use may, e.g., be constructedto release the active drug by controlling the dissolution and/or thediffusion of the active drug substance.

Dissolution or diffusion controlled release can be achieved byappropriate coating of a tablet, capsule, pellet, or granulateformulation of drugs, or by incorporating the drug into an appropriatematrix. A controlled release coating may include one or more of thecoating substances mentioned above and/or, e.g., shellac, beeswax,glycowax, castor wax, carnauba wax, stearyl alcohol, glycerylmonostearate, glyceryl distearate, glycerol palmitostearate,ethylcellulose, acrylic resins, dl-polylactic acid, cellulose acetatebutyrate, polyvinyl chloride, polyvinyl acetate, vinyl pyrrolidone,polyethylene, polymethacrylate, methylmethacrylate,2-hydroxymethacrylate, methacrylate hydrogels, 1,3 butylene glycol,ethylene glycol methacrylate, and/or polyethylene glycols. In acontrolled release matrix formulation, the matrix material may alsoinclude, e.g., hydrated methylcellulose, carnauba wax and stearylalcohol, carbopol 934, silicone, glyceryl tristearate, methylacrylate-methyl methacrylate, polyvinyl chloride, polyethylene, and/orhalogenated fluorocarbon.

A controlled release composition containing one or more of the drugs ofthe claimed combinations may also be in the form of a buoyant tablet orcapsule (i.e., a tablet or capsule that, upon oral administration,floats on top of the gastric content for a certain period of time). Abuoyant tablet formulation of the drug(s) can be prepared by granulatinga mixture of the drug(s) with excipients and 20-75% w/w ofhydrocolloids, such as hydroxyethylcellulose, hydroxypropylcellulose, orhydroxypropylmethylcellulose. The obtained granules can then becompressed into tablets. On contact with the gastric juice, the tabletforms a substantially water-impermeable gel barrier around its surface.This gel barrier takes part in maintaining a density of less than one,thereby allowing the tablet to remain buoyant in the gastric juice.

Liquids for Oral Administration

Powders, dispersible powders, or granules suitable for preparation of anaqueous suspension by addition of water are convenient dosage forms fororal administration. Formulation as a suspension provides the activeingredient in a mixture with a dispersing or wetting agent, suspendingagent, and one or more preservatives. Suitable suspending agents are,for example, sodium carboxymethylcellulose, methylcellulose, sodiumalginate, and the like.

Parenteral Compositions

The pharmaceutical composition may also be administered parenterally byinjection, infusion or implantation (intravenous, intramuscular,subcutaneous, or the like) in dosage forms, formulations, or viasuitable delivery devices or implants containing conventional, non-toxicpharmaceutically acceptable carriers and adjuvants. The formulation andpreparation of such compositions are well known to those skilled in theart of pharmaceutical formulation.

Compositions for parenteral use may be provided in unit dosage forms(e.g., in single-dose ampoules), or in vials containing several dosesand in which a suitable preservative may be added (see below). Thecomposition may be in form of a solution, a suspension, an emulsion, aninfusion device, or a delivery device for implantation or it may bepresented as a dry powder to be reconstituted with water or anothersuitable vehicle before use. Apart from the active drug(s), thecomposition may include suitable parenterally acceptable carriers and/orexcipients. The active drug(s) may be incorporated into microspheres,microcapsules, nanoparticles, liposomes, or the like for controlledrelease. The composition may include suspending, solubilizing,stabilizing, pH-adjusting agents, and/or dispersing agents.

The pharmaceutical compositions according to the invention may be in theform suitable for sterile injection. To prepare such a composition, thesuitable active drug(s) are dissolved or suspended in a parenterallyacceptable liquid vehicle. Among acceptable vehicles and solvents thatmay be employed are water, water adjusted to a suitable pH by additionof an appropriate amount of hydrochloric acid, sodium hydroxide or asuitable buffer, 1,3-butanediol, Ringer's solution, and isotonic sodiumchloride solution. The aqueous formulation may also contain one or morepreservatives (e.g., methyl, ethyl or n-propyl p-hydroxybenzoate). Incases where one of the drugs is only sparingly or slightly soluble inwater, a dissolution enhancing or solubilizing agent can be added, orthe solvent may include 10-60% w/w of propylene glycol or the like.

Controlled release parenteral compositions may be in form of aqueoussuspensions, microspheres, microcapsules, magnetic microspheres, oilsolutions, oil suspensions, or emulsions. Alternatively, the activedrug(s) may be incorporated in biocompatible carriers, liposomes,nanoparticles, implants, or infusion devices. Materials for use in thepreparation of microspheres and/or microcapsules are, e.g.,biodegradable/bioerodible polymers such as polygalactin, poly-(isobutylcyanoacrylate), poly(2-hydroxyethyl-L-glutamine). Biocompatible carriersthat may be used when formulating a controlled release parenteralformulation are carbohydrates (e.g., dextrans), proteins (e.g.,albumin), lipoproteins, or antibodies. Materials for use in implants canbe non-biodegradable (e.g., polydimethyl siloxane) or biodegradable(e.g., poly(caprolactone), poly(glycolic acid) or poly(ortho esters)).

Rectal Compositions

For rectal application, suitable dosage forms for a composition includesuppositories (emulsion or suspension type), and rectal gelatin capsules(solutions or suspensions). In a typical suppository formulation, theactive drug(s) are combined with an appropriate pharmaceuticallyacceptable suppository base such as cocoa butter, esterified fattyacids, glycerinated gelatin, and various water-soluble or dispersiblebases like polyethylene glycols. Various additives, enhancers, orsurfactants may be incorporated.

Percutaneous and Topical Compositions

The pharmaceutical compositions may also be administered topically onthe skin for percutaneous absorption in dosage forms or formulationscontaining conventionally non-toxic pharmaceutical acceptable carriersand excipients including microspheres and liposomes. The formulationsinclude creams, ointments, lotions, liniments, gels, hydrogels,solutions, suspensions, sticks, sprays, pastes, plasters, and otherkinds of transdermal drug delivery systems. The pharmaceuticallyacceptable carriers or excipients may include emulsifying agents,antioxidants, buffering agents, preservatives, humectants, penetrationenhancers, chelating agents, gel-forming agents, ointment bases,perfumes, and skin protective agents.

The emulsifying agents may be naturally occurring gums (e.g., gum acaciaor gum tragacanth).

The preservatives, humectants, penetration enhancers may be parabens,such as methyl or propyl p-hydroxybenzoate, and benzalkonium chloride,glycerin, propylene glycol, urea, etc.

The pharmaceutical compositions described above for topicaladministration on the skin may also be used in connection with topicaladministration onto or close to the part of the body that is to betreated. The compositions may be adapted for direct application or forapplication by means of special drug delivery devices such as dressingsor alternatively plasters, pads, sponges, strips, or other forms ofsuitable flexible material.

Dosages and Duration of the Treatment

It will be appreciated that the drugs of the combination may beadministered concomitantly, either in the same or differentpharmaceutical formulation or sequentially. If there is sequentialadministration, the delay in administering the second (or additional)active ingredient should not be such as to lose the benefit of theefficacious effect of the combination of the active ingredients. Aminimum requirement for a combination according to this description isthat the combination should be intended for combined use with thebenefit of the efficacious effect of the combination of the activeingredients. The intended use of a combination can be inferred byfacilities, provisions, adaptations and/or other means to help using thecombination according to the invention.

Although the active drugs of the present invention may be administeredin divided doses, for example two or three times daily, a single dailydose of each drug in the combination is preferred, with a single dailydose of all drugs in a single pharmaceutical composition (unit dosageform) being most preferred.

The term “unit dosage form” refers to physically discrete units (such ascapsules, tablets, or loaded syringe cylinders) suitable as unitarydosages for human subjects, each unit containing a predeterminedquantity of active material or materials calculated to produce thedesired therapeutic effect, in association with the requiredpharmaceutical carrier.

Administration can be one to several times daily for several days toseveral years, and may even be for the life of the patient. Chronic orat least periodically repeated long-term administration will beindicated in most cases.

Additionally, pharmacogenomic (the effect of genotype on thepharmacokinetic, pharmacodynamic or efficacy profile of a therapeutic)information about a particular patient may affect the dosage used.

Except when responding to especially impairing AD disease cases wherehigher dosages may be required, the preferred dosage of each drug in thecombination usually lies within the range of doses not above thoseusually prescribed for long-term maintenance treatment or proven to besafe in phase 3 clinical studies.

One remarkable advantage of the invention is that each compound may beused at low doses in a combination therapy, while producing, incombination, a substantial clinical benefit to the patient. Thecombination therapy may indeed be effective at doses where the compoundshave individually no substantial effect. Accordingly, a particularadvantage of the invention lies in the ability to use sub-optimal dosesof each compound, i.e., doses which are lower than therapeutic dosesusually prescribed, preferably ½ of therapeutic doses, more preferably⅓, ¼, ⅕, or even more preferably 1/10 to 1/100 of therapeutic doses. Atsuch sub-optimal dosages, the compounds alone would be substantiallyinactive, while the combination(s) according to the invention are fullyeffective.

A preferred dosage corresponds to amounts from 1% up to 50% of thoseusually prescribed for long-term maintenance treatment, for instancefrom 1% up to 10%.

Specific examples of dosages are provided below:

-   -   Zonisamide orally from 1 to 200 mg per day,    -   Dyphylline orally from 6 to 300 mg per day divided in two or        three doses,    -   Tadalafil orally from 0.05 to 2.5 mg per day,    -   acamprosate orally from 1 to 50 mg per day,    -   cinacalcet orally from 0.3 to 15 mg per day,    -   terbinafine orally from 2.5 to 75 mg per day,    -   cilostazol orally from 1 mg to 50 mg per day,    -   baclofen orally from 0.4 to 40 mg per day divided in two or        three doses,    -   phenformin orally from 0.5 to 25 mg per day,    -   amlodipine orally from 0.05 to 5 mg per day,    -   sulfisoxazole orally from 0.4 to 4 g per day divided in 6 to 4        doses.

Examples of dosages for combined therapies are provided below:

-   -   dasatinib orally from about 1 to 10 mg per day and acamprosate        orally from about 7 to 70 mg three times daily,    -   aztreonam orally from about 20 to 1400 mg per day in 4 divided        doses and tiagabine orally from about 0.3 to 3 mg per day,    -   chlorzoxazone orally from about 5 to 50 mg 3 or 4 times per day        and tadalafil orally from about 0.05 to 0.5 mg per day,    -   chlorzoxazone orally from about 5 to 50 mg 3 or 4 times per day        and cilostazol orally from about 1 to 10 mg per day,    -   chlorzoxazone orally from about 5 to 50 mg 3 or 4 times per day        and terbinafine orally from about about 2.5 to 25 mg once or        twice daily,    -   chlorzoxazone orally from about 5 to 50 mg 3 or 4 times per day        and dasatinib orally from about 1 to 10 mg per day,    -   dasatinib orally from about 1 to 10 mg per day and terbinafine        orally from about 2.5 to 25 mg once or twice daily,    -   cinacalcet orally from about 0.3 to 3 mg per day and acamprosate        orally from about 7 to 70 mg three times daily,    -   aztreonam orally from about 20 to 1400 mg per day in 4 divided        doses and vigabatrin orally from about 20 to 200 mg once or        twice per day,    -   topiramate orally from about 2 to 60 mg per day and dyphylline        orally from about 6 to 60 mg per day in two or three divided        doses.

It will be understood that the amount of the drug actually administeredwill be determined by a physician, in the light of the relevantcircumstances including the condition or conditions to be treated, theexact composition to be administered, the age, weight, and response ofthe individual patient, the severity of the patient's symptoms, and thechosen route of administration. Therefore, the above dosage ranges areintended to provide general guidance and support for the teachingsherein, but are not intended to limit the scope of the invention.

The following examples are given for purposes of illustration and not byway of limitation.

EXAMPLES

I. Compounds Prevent the Toxicity of Aβ₂₅₋₃₅ Peptide

In this first series of experiments, candidate compounds have beentested for their ability to prevent or reduce the toxic effects ofAβ₂₅₋₃₅ peptide.

In AD, the protein forms aggregates of insoluble β-pleated sheets offibrillar Abeta protein (amyloid). The conformational change fromsoluble to fibrillar forms seems to be a spontaneous event that isincreased with higher concentrations of Abeta, so any production oflarger amounts of Abeta than normal (or production of the larger, lesssoluble forms of Abeta) will tend to increase plaque formation. Once theAbeta plaque has started to form, other molecules can interact with thenascent plaque to produce eventually the mature plaque with itsassociated areas of neuronal cell death. Considering this, we haveevaluated the effects of our candidate drugs on the viability of thecells exposed to the amyloid β protein.

Cell Culture

Primary rat cortical neurons are cultured as described by Singer et al.,1999. Briefly pregnant female rats of 15 days gestation are killed bycervical dislocation (Rats Wistar; Janvier) and the fetuses removed fromthe uterus. The cortex are removed and placed in ice-cold medium ofLeibovitz (L15; Invitrogen) containing 1% of Penicillin-Streptomycin(PS; Invitrogen) and 1% of bovine serum albumin (BSA; Sigma). Cortex aredissociated by trypsinisation for 20 min at 37° C. (Trypsin EDTA 1×;Invitrogen) diluted in PBS without calcium and magnesium. The reactionis stopped by the addition of Dulbecco's modified Eagle's medium (DMEM;Invitrogen) containing DNAase I grade II (0.1 mg/ml; Roche Diagnostic)and 10% of foetal calf serum (FCS; Invitrogen). Cells are thenmechanically dissociated by 3 passages through a 10 ml pipette. Cellsare then centrifuged at 180×g for 10 min at 10° C. The supernatant isdiscarded and the cells of pellet are re-suspended in a defined culturemedium consisting of Neurobasal (Invitrogen) supplemented with B27 (2%;Invitrogen), L-glutamine (0.2 mM; Invitrogen), 1% of PS solution and 10ng/ml of Brain-derived neurotrophic factor (BDNF, Pan Biotech). Viablecells are counted in a Neubauer cytometer using the trypan blueexclusion test. Cells are seeded at a density of 30 000 cells/well in 96well-plates (wells are pre-coated with poly-L-lysine (10 μg/ml; Sigma)and are cultured at 37° C. in a humidified air (95%)/CO2 (5%)atmosphere.

After 6 days of culture, cells are incubated with drugs (5concentrations). After 1 hour, cells are intoxicated by 20 μM ofbeta-amyloïd (25-35; Sigma) in defined medium without BDNF but togetherwith drugs. Cortical neurons are intoxicated for 2 days. BDNF (10 ng/ml)is used as a positive (neuroprotective) control. Two independentcultures are performed per condition, 6 wells per condition.

Neurites Length Quantification

Cells are fixed with a cool solution of ethanol (95%) and acetic acid(5%) for 10 min. After permeabilization with 0.1% of saponin, cells areblocked for 2 h with PBS containing 10% goat serum. Cells are thenincubated with monoclonal antibody directed against the microtubuleassociated protein 2 (MAP-2; Sigma). This antibody reveals specificallycell bodies and neurites. The secondary antibody used is an Alexa Fluor488 goat anti-mouse IgG (Molecular probe). Nuclei of neurons arerevealed by a fluorescent dye (Hoechst solution, SIGMA). Twenty picturesare taken per well, using InCell Analyzer™ 1000 (GE Healthcare) atmagnification 20×. All images are taken in the same conditions. Neuriteslength is quantified using Developer software (GE Healthcare).

Results

The results are presented in FIGS. 1A, 1B, and 2. These results areextracted from two independent cultures, 6 wells per condition. Allvalues are expressed as mean±s.e. mean. A bilateral Student's t testanalysis is performed on raw data. Results are expressed in percentageof neurites length, compared to the control (vehicle).

Drugs were incubated with rat primary cortical neurons one hour beforeAbeta₂₅₋₃₅ 20 μM intoxication that lasts 2 days (40).

Two days after this incubation the network of neurites length wasquantified, reflecting axonal cell growth. The results clearlydemonstrate a neuroprotective effect of the tested drugs of theinvention against Abeta₂₅₋₃₅ intoxication (FIGS. 1A, 1B, and FIG. 2).

II. Compounds and Combinations Thereof Prevent the Toxicity of HumanAβ₁₋₄₂ Peptide

In this further series of experiments, candidate compounds have beentested for their ability to prevent or reduce the toxic effects of humanAβ₁₋₄₂. Aβ₁₋₄₂ is the full length peptide that constitutes aggregatesfound in biopsies from human patients afflicted with AD. The drugs arefirst tested individually, followed by assays of their combinatorialaction. The effect is determined on various cell types, to furtherdocument the activity of the compounds.

II.1. Protection Against the Toxicity of Human Aβ₁₋₄₂ Peptide in RatPrimary Cortical Neuron Cells

Test Compound and Human Amyloid-β1-42 Treatment

Primary rat cortical neurons are cultured as described previously.Briefly, Aβ₁₋₄₂ peptide was reconstituted in define culture medium at 40μM (mother solution) and was slowly shaked at +37° C. for 3 days indark. The control medium was prepared in the same conditions.

After 3 days, the solution was used on primary cortical neurons asfollows:

After 10 days of neuron culture, drug was solved in culture medium(+0.1% DMSO) and then pre-incubated with neurons for 1 hour before theAβ₁₋₄₂ application (in a final volume per culture well of 100 μl). Onehour after drug incubation, 100 μl of Aβ₁₋₄₂ peptide was added to afinal concentration of 10 μM diluted in presence of drug, in order toavoid further drug dilutions. Cortical neurons were intoxicated for 24hours. Three separate cultures were performed per condition, 6 wells percondition.

BDNF (50 ng/ml) and Estradiol-β (100 and 150 nM) were used as positivecontrol and reference compounds respectively. Three separate cultureswill be performed per condition, 12 wells per condition.

Organization of Culture Plates

Estradiol-β at 100 and 150 nM were used as reference test compound andBDNF at 50 ng/ml was used as a positive control. Estradiol-β and BDNFwere solved in culture medium and pre-incubated for 1 h before theamyloid-β₁₋₄₂ application.

The following conditions were assessed:

-   -   1 CONTROL PLAQUE: 12 wells/condition    -   Negative Control: medium alone +0.1% DMSO    -   Intoxication: amyloid-β₁₋₄₂ (10 μM) for 24 h    -   Positive control: BDNF (50 ng/ml) 1 hr followed by amyloid-β₁₋₄₂        (10 μM) for 24 h    -   Reference compound: Estradiol (150 nM) 1 hr followed by        amyloid-β₁₋₄₂ (10 μM) for 24 h.    -   DRUG PLATE: 6 wells/condition    -   Negative Control: medium alone+0.1% DMSO    -   Intoxication: amyloid-β₁₋₄₂ (10 μM) for 24 h    -   Drug 1: Drug 1—1 hr followed by amyloid-β₁₋₄₂ (10 μM) for 24 h    -   Drug 2: Drug 2—1 hr followed by amyloid-β₁₋₄₂ (10 μM) for 24 h

Lactate Dehydrogenase (LDH) Activity Assay

24 hours after intoxication, the supernatant was taken off and analyzedwith Cytotoxicity Detection Kit (LDH, Roche Applied Science, ref:11644793001, batch: 11800300). This colorimetric assay for thequantification of cell toxicity is based on the measurement of lactatedehydrogenase (LDH) activity released from the cytosol of dying cellsinto the supernatant.

Data Processing

All values are expressed as mean±s.e. mean of the 3 cultures (n=6 percondition). Statistic analyses were done on the different conditions(ANOVA followed by the Dunnett's test when it was allowed, Statviewsoftware version 5.0).

Results

The results are presented in table 4 below and are exemplified in FIGS.3A, 3B, and 3C. These results clearly demonstrate a substantive effectof the drugs of the invention on Aβ₁₋₄₂-intoxicated neural cells.

TABLE 4 Protective effect on Aβ₁₋₄₂ toxicity DRUG NAME in neuronal cellsBaclofen (+/−) + Cinacalcet + Phenformin + Sulfisoxazole + Tadalafil +Zonisamide +

II.2. Protection Against the Toxicity of Human Aβ₁₋₄₂ Peptide on HumanBrain Microvascular Endothelial Cells

Ultrastructural studies have shown that brain microvessels are closelyassociated with β-amyloid plaques, and that Alzheimer's disease braincapillaries contain preamyloid deposits (42). Damage to the vasculatureresulting from Abeta deposition can result in a reduction of cerebralblood flow (43). Moreover, Abeta peptides have been shown to be potentinhibitors of angiogenesis in vitro and in vivo (41). Aβ1-42 is the fulllength peptide that constitutes aggregates found in biopsies from humanpatients that suffered from AD. To be the closest as possible of thehuman disease, the protection afforded by candidate compounds towardAβ1-42 was assessed.

We then chose to use Human Brain Microvascular Endothelial Cells (HBMEC)to further illustrate the protective effect of our compounds against theAβ1-42 peptide injury. This model has been previously used to study theanti-angiogenic properties of mutant forms of Abeta peptide.

Human brain microvascular endothelial cerebral cells (HBMEC, ScienCellRef: 1000, frozen at passage 10) were rapidly thawed in a waterbath at+37° C. The supernatant was immediately put in 9 ml Dulbecco's modifiedEagle's medium (DMEM; Pan Biotech ref: P04-03600) containing 10% offoetal calf serum (FCS; GIBCO ref 10270-106). Cell suspension wascentrifuged at 180×g for 10 min at +4° C. and the pellets were suspendedin CSC serum-free medium (CSC serum free, Cell System, Ref:SF-4Z0-500-R, Batch 51407-4) with 1.6% of Serum free RocketFuel (CellSystem, Ref: SF-4Z0-500-R, Batch 54102), 2% of Penicillin 10.000 U/mland Streptomycin 10 mg/ml (PS; Pan Biotech ref: P06-07100 batch133080808) and were seeded at the density of 20 000 cells per well in 96well-plates (matrigel layer biocoat angiogenesis system, BD, Ref 354150,Batch A8662) in a final volume of 100 μl. On matrigel support,endothelial cerebral cells spontaneously started the process ofcapillary network morphogenesis (41). Three separate cultures wereperformed per condition, 6 wells per condition.

Candidate Compounds and Human Amyloid-β₁₋₄₂ Treatment

Briefly, Aβ1-42 peptide (Bachem, ref: H1368 batch 1010533) wasreconstituted in define culture medium at 20 μM (mother solution) andwas slowly shacked at +37° C. for 3 days in dark. The control medium wasprepared in the same conditions. After 3 days, this human amyloidpeptide was used on HBMEC at 2.5 μM diluted in control medium (optimalincubation time). The Aβ1-42 peptide was added 2 hours after HBMECseeding on matrigel for 18 hours incubation.

One hour after HBMEC seeding on matrigel, test compounds and VEGF-165were solved in culture medium (+0.1% DMSO) and then pre-incubated withHBMEC for 1 hour before the Aβ₁₋₄₂ application (in a final volume perculture well of 100 μl). One hour after test compounds or VEGFincubation (two hours after cell seeding on matrigel), 100 μl of Aβ₁₋₄₂peptide was added to a final concentration of 2.5 μM diluted in controlmedium in presence of test compounds or VEGF (in a 200 μl totalvolume/well), in order to avoid further drug dilutions.

Organization of Culture Plates

VEGF-165, known to be a pro-angiogenic isoform of VEGF-A, was used forall experiments in this study as reference compound. VEGF-165 is one ofthe most abundant VEGF isoforms involved in angiogenesis. VEGF was usedas reference test compound at 10 nM.

The following conditions were assessed:

-   -   Negative Control: medium alone +0.1% DMSO    -   Intoxication: amyloid-β₁₋₄₂ (2.5 μM) for 18 h    -   Positive control: VEGF-165 (10 nM) (1 reference        compound/culture) 1 hr before the Aβ₁₋₄₂ (2.5 μM) addition for a        18 h incubation time.    -   Test compounds: Test compound 1 hr before the Aβ₁₋₄₂ (2.5 μM)        addition for a 18 h incubation time.

Capillary Network Quantification

Per well, 2 pictures with 4× lens were taken using InCell Analyzer™ 1000(GE Healthcare) in light transmission. All images were taken in the sameconditions. Analysis of the angiogenesis networks was done usingDeveloper software (GE Healthcare). The total length of capillarynetwork was assessed.

Data Processing

All values are expressed as mean±s.e. mean of the 3 cultures (n=6 percondition). Statistic analyses were done on the different conditionsperforming an ANOVA followed by the Dunnett's test when it was allowed(Statview software version 5.0). The values (as %) inserted on thegraphs show the amyloid toxicity evolution. Indeed, the amyloid toxicitywas taken as the 100% and the test compound effect was calculated as a %of this amyloid toxicity.

Results

The results are shown in Table 5.

TABLE 5 Protective effect in Aβ₁₋₄₂ intoxicated DRUG NAME HBMEC cellsBaclofen (+/−) + Sulfisoxazole + Terbinafine + Zonisamide + Phenformin +

These results clearly show a protective effect of single drugs on humancells.

II.3. Effect of Combined Therapies on the Toxicity of Human Aβ₁₋₄₂Peptide on Human HBMEC Cells and on Rat Primary Cortical Neurons

We have also tested the efficacy of drug combinations of the inventionin a human and rat system. The protocols used in these experiments arethe same as described in sections II.1 and II.2 above.

Results

The following drug combinations are tested on human brain microvascularendothelial cells and on rat primary cortical neuron cells:

-   -   phenformin and zonisamide,    -   zonisamide and sulfisoxazole,    -   acamprosate and zonisamide,    -   zonisamide and dyphylline,    -   zonisamide and argatroban,    -   zonisamide and cilostazol,    -   phenformin and acamprosate,    -   phenformin and sulfisoxazole,    -   acamprosate and sulfisoxazole,    -   phenformin and dyphylline,    -   phenformin and cilostazol,    -   phenformin and tadalafil, and    -   zonisamide and terbinafine.

All of the tested drug combinations give protective effect againsttoxicity of human Aβ₁₋₄₂ peptide in both models, as shown in Table 6below.

TABLE 6 Protective effect on Protective effect in Aβ₁₋₄₂ toxicity Aβ₁₋₄₂intoxicated DRUG NAME in neuronal cells HBMEC cells phenformin andzonisamide + + zonisamide and sulfisoxazole + + acamprosate andzonisamide + + zonisamide and dyphylline + + zonisamide andargatroban + + zonisamide and cilostazol + + phenformin andacamprosate + + phenformin and sulfisoxazole + + acamprosate andsulfisoxazole + + phenformin and dyphylline + + phenformin andcilostazol + + phenformin and tadalafil + + zonisamide and terbinafine ++

III. In Vivo Activity

Compounds and their combinations are tested in an in vivo model ofAlzheimer disease. Overexpression of Alzheimer's disease-linked mutanthuman amyloid beta protein precursor (APP) transgenes has been the mostreliable means of promoting deposition of Abeta in the brains oftransgenic mice that served as AD disease models in numerous studies. Asthey age, these mutant APP mice develop robust amyloid pathology andother AD-like features, including decreased synaptic density, reactivegliosis, and some cognitive deficits. Many mutant APP mouse models showlittle evidence of overt neuronal loss and neurofibrillary tangle (NFT)pathology. Mice hemizygous for this BRI-Abeta42 transgene are viable andfertile with a normal lifespan. Transgenic BRI-Abeta42 mRNA is expressedin a pattern characteristic of the mouse prion protein promoter; highesttransgene expression levels are detected in the cerebellar granule cellsand hippocampus, followed by the cortex, pons, thalamus, and midbrain.In the transgenic fusion protein, Abeta1-42 is fused to the C terminusof the BRI protein at the furin-like cleavage site such that cleavageresults in efficient Abeta1-42 secretion into the lumen or extracellularspace. Therefore, these mice specifically express the Abeta1-42 isoform.Hemizygous BRI-Abeta42 mice accumulate detergent-insoluble amyloid-betawith age and develop cored plaques in the cerebellum at as early as 3months of age.

Development of forebrain pathology occurs later, extracellular Abetaplaques are not present consistently in the hippocampus andentorhinal/piriform cortices until 12 months of age. Amyloid betadeposits (cored plaques) can be observed as early as 3 months inmolecular layer of cerebella of transgenic mice and becoming morepronounced with age; occasional extracellular plaques are seen in theentorhinal/piriform cortices and hippocampus at 6 months of age, butaren't consistently found until >12 months of age. Oldest mice showwidespread pathology with cored and diffuse plaques in cerebellum,cortex, hippocampus, and olfactory bulb. Extracellular amyloid plaquesshow dense amyloid cores with radiating fibrils; many bundles ofdystrophic neurites are observed at the periphery of these plaques.Reactive gliosis is associated with plaques.

Drug Treatments

The transgenic Tg (Prnp-ITM2B/APP695*42) A12E mc mice (31) has beenobtained from Jackson Laboratory(http://jaxmice.jax.org/strain/007002.html). Mice founder with thehighest Abeta42 plasma levels, line BRI-Abeta42A (12e), have beenmaintained on a mixed B6C3 background. Adult male transgenic mice havefree access to food and water. In accord with an approved theInstitutional Animal Care and Use Committee protocol, mice have beenweighed and injected i.p. or force fed once daily for 10 to 20consecutive weeks with either a control solution (placebo) or PXT drugs,prepared at different doses.

Survival Analysis

Survival rates have been analyzed using Kaplan-Meier methods. Holm-Sidakmethods (post hoc) have been used for all pairwise multiple comparisontests. The extraneous deaths are censored. All comparisons have beenmade between littermates to limit any potentially confounding effectsfrom background strain differences.

Behavioural Tests

Behavioural tests were designed and conducted according to the methodspublished by several authors (32-35).

Spatial Learning and Memory in the Morris Water Maze (MWM)

This experiment is performed in a circular pool, 90 cm in diameter, madeof white plastic and filled with milky colored water. An escapeplatform, 8 cm in diameter, made of clear plastic was submerged 0.5 cmunder the water level. Visual clues are provided by differentgeometrical forms printed in A4-sized letters and placed on the foursurrounding walls (distance from the pool was from 50 to 70 cm). Eachmouse has been given four trials daily (5- to 7-minute interval betweentrials, a total of 16 trials) for 4 days. Each trial has been performedfrom one of four different starting points. The movement of the mice ismonitored using Videotrack Software (View Point). The time taken tolocate the escape platform (escape latency; up to 60 seconds) has beendetermined. After locating the platform the mouse has been allowed tosit on it for 15 seconds. Mice who failed to find the platform within 60seconds have been guided to it and allowed to stay on it for 15 seconds.A latency of 60 seconds is entered into the record for such anoccurrence. All four trials per day have been averaged for statisticalanalysis, except for the first trial on day 1. On day 9 (5 days afterthe last training) mice have been subjected to a 60-second probe trialin which the platform is removed and the mice are allowed to search forit. The time that each animal spent in each quadrant has been recorded(quadrant search time). Several groups of male mice have been used at 3,7, 10, and 12 months. The some few mice have showed freezing behaviour(e.g., lying motionless in the water and refusing to swim) that stronglyinterfered with the test, these animals have been excluded from the dataanalysis. All behavioural tests are conducted under a quiet andlight-reduced environment.

Working Memory Test in Radial Arm Water Maze

This cognitive-based sensitive measure of working memory has beenobtained with the help of the apparatus consisted of a 100 cm-diameterwaterfilled pool (also used for the Morris water maze and PlatformRecognition tasks) fitted with an aluminium insert to create sixradially-distributed swim arms. Testing consists of five, 1-min trialsper daily session, for 9-12 consecutive days. At the start of eachsession, a clear submerged platform is positioned at the end of one ofthe six swim arms (randomly-selected, changed daily). For each of thefirst four acquisition trials, the animal is placed into one of thenon-platform containing arms (randomized sequence) and allowed to searchfor the platform. During the 60 s trial, each time the animal entersanother non-platform containing arm, it is gently returned to itsstarting location and an error recorded. After the fourth trial, theanimal is allowed to rest for 30 min, followed by a fifth (retention)trial, which originates in the final non-platform containing swim arm.The number of errors (incorrect arm choices) and escape latency (time toreach platform, maximum 60 s) are recorded for each trial.

Spatial Reference Learning and Memory in Circular Platform Test

This cognitive-based task test is performed with the help of theapparatus that consists of a 69 cm-diameter circular platform having 16“escape” holes spaced equidistantly around the circumference. An escaperefuge is installed beneath one of the holes, and a black curtain, onwhich are placed various visual cues, encircles the platform. The animalis placed in the center of the platform at the start of a single, 5 mintrial and aversive stimuli (bright lights, fan wind) are presented. Thetotal number of errors (head-pokes into non-escape holes) and escapelatency (time to reach escape hole) are recorded.

Recognition Ability in Platform Recognition Test

This cognitive-based search task evaluates object identification andrecognition ability. The target object consists of a 9 cm-diametercircular platform fitted with a 10 cm×40 cm black ensign, which ispositioned 0.8 cm above the surface of the water in a 100 cm-diametercircular pool. Testing consists of four 60 s trials per day on each offour consecutive days. On each day, the target object is placed into adifferent quadrant of the pool for each trial, and the animal isreleased at the same location along the circumference of the pool forall four trials. The total latency (maximum 60 s) is recorded for eachtrial.

Modified Irwin Examination

A comprehensive screen, modified from Irwin is used to determine whetherany of the mice exhibited physiological, behavioural, or sensorimotorimpairments related to their genotype. To explore motor skills,coordination, and muscle strength, the mice are placed on a wire thatwas tightened between two 30-cm-high columns and their ability tobalance on the wire is assessed. In addition, their ability to grasp andhang on the wire with all four paws for at least 5 seconds and to climbback on the wire is determined.

Quantification of Vascular Amyloid Deposition

For quantification of cerebral amyloid angiopathy (CAA), 5 μmparaffin-embedded sections at 30 μm intervals through the parietal orcerebellar cortex leptomeninges are immunostained with biotinylated-Ab9antibody (anti-Aβ1-16, 1:500) overnight at 4° C. (n=5-7 mice pergenotype at each age group, n=6 sections per mouse). Positively stainedblood vessels are visually assessed using modified Vonsattel's scoringsystem (36) The CAA severity score is calculated by multiplying thenumber of CAA vessels with the CAA severity grade.

Histology: Immunohistochemistry and Immunofluorescence

Tg and WT mice from 3 to 12 months are anesthetized and transcardiallyperfused sequentially with 0.9% NaCl and 4% paraformaldehyde in 0.1mol/L phosphate buffered saline (PBS) (pH 7.4) or 10% formalin and 4%paraformaldehyde in 0.1 mol/L PBS (pH 7.4). Brains and spinal cords areremoved and stored in 4% paraformaldehyde. Some samples are embedded inparaffin and cut on a sliding microtome at a thickness of 10 μm.Cryosections (14 μm) are cut on a cryostat and mounted on chromealum-coated slides. Endogenous peroxidase is quenched by treating thesection with methanol containing 0.3% H2O2 for 30 minutes. Sections areblocked in 10% horse serum. Primary antibodies are used and incubatedovernight at 4° C. in the presence of 1% horse serum. All secondarybiotinylated or fluorescein-, Texas Red-, and AMCA-coupled antibodies,fluorochromes, ABC-kit, and 3,3′-diaminobenzidine as chromogen forperoxidase activity are from Vector Laboratories. Incubation with thesecondary antibody is held at room temperature for 1 hour. All washingsteps (3-10 minutes) and antibody dilution are performed usingphosphate-buffered saline (0.1 mol/L PBS, pH 7.4) or Tris-bufferedsaline (0.01 mol/L Tris, 0.15 mol/L NaCl, pH 7.4). Incubation with theABC complex and detection with 3,3′-diaminobenzidine is carried outaccording to the manufacturer's manual. Hematoxylin counterstaining isperformed according to standard procedures. A minimum of three mice pergenotype, age, and sex is used for each determination (37).

Preparation of Brain Extracts

Brains are rapidly harvested over ice between 90 and 120 min after thefinal injection and frozen to −80° C. The right cerebral hemisphere fromeach mouse is weighed after freezing. Analysis of hemisphere mass bymedian absolute deviation allows us to exclude samples that are beyond 4median absolute deviations from the rest of the set. Cerebralhemispheres are homogenized, and cell lysates containing whole proteinare prepared according to the manufacturer's instructions for enzymaticassay kits (R&D Systems, Inc.). In brief, the brain cortices arehomogenized in 800 μl of low salt containing 1× extraction buffer (R&Dkit) and incubated on ice for 10 min. The homogenates are thencentrifuged at 13,000 g for 15 min at 4° C. The protein concentration ineach sample is estimated according to biuret-derived assay (Pierce).Levels of APP, Aβ40, and Aβ42 are measured by Western immunoblotting andsandwich ELISA techniques, respectively, as described (28). In addition,activities of α, β-, and γ-secretases may be measured from the sameextracts.

Assay of Levels of Total APP in Mouse Cerebral Cortex Extracts

An equal-protein amount of brain extracts is loaded in each gel, 30 μgper lane per sample. Each gel contained eight treatments: control; drug17.5 mg/kg dose; and drug 2 in several doses. To minimize intra-gelvariation, each gel contained three sets of all treatment groups. Eachblot is probed with 22C11 antibody. Each blot is also probed with theβ-actin antibody for normalization to transfer efficiency. The intensityof APP band signal is normalized with that of β-actin. Two sample“controls” are loaded in each gel/blot to test for blot to blotvariation. Analysis of blots is performed in two ways: blot wise (n=3),to test for gel to gel variation; and combined blots (n=9 or 10) asdescribed (38-39). Blot-wise analysis with n=3 shows the same trend asthe final analysis with n=9 or 10 does. Results of the combined analysisare presented.

Aβ Sandwich ELISA

For brain Aβ ELISAs, forebrain and hindbrain Aβ levels are determinedindependently, and the olfactory bulb is excluded from analysis. Forplasma Aβ analysis, blood is collected in EDTA-coated tubes aftercardiac puncture. Blood samples are centrifuged at 3000 rpm for 10 minat 4° C., and the plasma is aliquoted and stored at −80° C. until used.Aβ levels are determined by end-specific sandwich ELISAs using Ab9(anti-Aβ1-16 Ab) as the capture Ab for Aβ40, 13.1.1-HRP (anti-Aβ35-40Ab) as the detection Ab for Aβ40, 2.1.3 (anti-Aβ35-42 Ab) as the captureAb for Aβ42, and Ab9-HRP as the detection Ab for Aβ42 (n=5-7 mice pergenotype at each age group). Aβ levels are normalized to the previousresults using the same sets of mice as internal controls to minimizepotential ELISA variability, as described (28).

Western Blotting

Snap-frozen forebrain samples are homogenized inradioimmunoprecipitation assay (RIPA) buffer (Boston BioProducts,Worcester, Mass.) with 1% protease inhibitor mixture (Roche). Thehomogenate is centrifuged at 100,000×g for 1 h at 4° C. Proteinconcentration in supernatants is determined using the BCA protein assay(Pierce). Protein samples (20 μg) are run on Bis-Tris 12% XT gels orBis-Tris 4-12% XT gels (Bio-Rad, Hercules, Calif.) and transferred to0.2 μm nitrocellose membranes. Blots are microwaved for 2 min in 0.1 MPBS twice and probed with Ab 82E1 (anti-Aβ1-16, 1:1000; IBL, Gunma,Japan) and anti-APP C-terminal 20 amino acids (1:1000) as described(28). Blots are stripped and reprobed with anti β-actin (1:1000; Sigma)as a loading control. Relative band intensity is measured using ImageJsoftware.

Quantification of Parenchymal Amyloid Deposition

Hemibrains are immersion fixed in 10% formalin and processed forparaffin embedding. Brain tissue sections (5 μm) were immunostained withanti-total Aβ antibody (Ab). Sections are counterstained withhematoxylin. Six sections per brain through the hippocampus, piriformcortex (bregma, −1.70 to −2.80 mm), or cerebellum (paraflocculus, crusansiform, and simple lobules; bregma, −5.40 to −6.36 mm) are used forquantification (n=5-7 mice per genotype at each age group). The Aβplaque burden is determined using MetaMorph software (Molecular Devices,Palo Alto, Calif.). For quantification of cored plaques, serial sectionsof those analyzed for Aβ burden are stained with thioflavine S (ThioS),and the number of ThioS-positive plaques in the hippocampus,entorhinal/piriform cortex, or the cerebellum is counted. All of theabove analyses are performed in a blinded manner.

Statistical Analysis of In Vivo Data

Results from all experiments are analyzed with STATISTICA 8.0(Statsoft).

Aβ levels, amyloid plaque burden, and CAA severity are analyzed by usingANOVA with the post hoc Holm-Sidak multiple comparison test ortwo-tailed Student's t test. If the data set does not meet theparametric test assumptions, either the Kruskal-Wallis test followed bythe post hoc Dunn's multiple comparison or the Mann-Whitney rank sumtest is performed. To test whether the Aβ levels in the bitransgenicmice were consistent with an additive sum of Aβ levels in the singletransgenic littermates, a multiple linear regression with no intercepttest is used. All comparisons are made between littermates.

Drug response modelling is done excluding the control (0 mg/kg) samples.ED50 corresponds to the dose (mg/kg) required to induce a 50% of maximaldrug-induced response in the experiments. It is calculated using theHill equation model for the log of ED50.

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The invention claimed is:
 1. A method of treating Alzheimer's disease,the method comprising administering to a subject in need thereof asynergistically effective amount of zonisamide and tadalafil, or saltsor sustained release formulations thereof.
 2. The method of claim 1,wherein the synergistically effective amount of zonisamide andtadalafil, or salts or sustained release formulations thereof, isformulated with a pharmaceutically acceptable carrier or excipient. 3.The method of claim 1, wherein the synergistically effective amount ofzonisamide and tadalafil, or salts or sustained release formulationsthereof, is repeatedly administered to the subject.
 4. The method ofclaim 1, wherein the compounds are administered separately.
 5. Themethod of claim 1, wherein the compounds are administered sequentially.6. The method of claim 1, wherein the compounds are administeredsimultaneously.